some current perspectives on chemical …...this limited list of known carcinogenic chemicals ex...

[CANCER RESEARCH 38. 1479-1496, June 1978]0008-5472/78/0038-0000$02.00.

Some Current Perspectives on Chemical Carcinogenesis in Humans andExperimental Animals: Presidential Address1

Elizabeth C. Miller

McArdle Laboratory lor Cancer Research, University of Wisconsin Medical Center, Madison, Wisconsin 53706

I am very honored to have the opportunity to discusssome aspects of chemical carcinogenesis with you tonight.Those of you who know my husband, James A. Miller, andme could not have been surprised at my choice of thissubject, for we have spent an exciting 35 years together intrying to ferret out some of the properties of chemicalcarcinogens and the changes that they cause in their targettissues. When we started our work together in 1942, chemical carcinogenesis was a rather limited field that hadattracted only a small number of investigators. During theintervening years interest in chemical carcinogenesis hasgrown markedly and, like the scientific disciplines of organic chemistry, biochemistry, and molecular biology, onwhich it depends, has grown greatly in its sophistication.

Knowledge in chemical carcinogenesis now spans manydisciplines and is so large a subject that many areas can beconsidered here only briefly or not at all. Similarly, in manycases references to reviews have been substituted forreferences to the original literature in order to keep thebibliography within manageable limits.

Environmental Chemical Carcinogens for the Human

The great interest in chemical carcinogenesis amongboth scientific and lay persons is based in part on theconclusion of epidemiologists, starting with Higginson (78)in 1969, that 60 to 90% of human cancers have importantenvironmental factors in their etiologies. This deduction isbased primarily on the large differences in incidences ofspecific cancers, usually measured by mortality figures,from country to country and even within countries (see alsoRefs. 41 and 42). As shown by Haenszel and his collaborators (65, 66) and others, these differences in geographicincidences are not primarily genetically determined. Thus,the cancer patterns for migrants from one country toanother, and especially those of their children, generallychange from those characteristic for inhabitants of themother country toward those characteristic of the inhabitants of their new country.

Other than skin cancer, for which solar UV is an importantcausative factor (44, 212), emphasis has been placed onenvironmental chemicals as major factors in the causationof human cancer. This emphasis has resulted from the lackof definitive data on the roles of infectious viruses in thecausation of human cancers (70),2 the indication that ioniz

ing radiations play only a relatively minor role in the overallcancer incidences (88), the fact that over a dozen specificchemicals have been identified as causes of some humancancers (Table 1), and the conclusion that a high proportionof all human lung cancers is associated with cigarettesmoking (191). In addition to the chemicals generally recognized as carcinogens in humans as a result of industrial,medical, and societal exposures, a number of other chemicals in the environment, such as aflatoxin B, and certain N-nitrosamines and N-nitrosamides, are strongly suspectedof causing cancers in humans (13, 84, 119, 216, 231). Itappears very likely that additional chemical carcinogens ofboth natural and synthetic origin will be identified as causesof human cancer.

1 Presented on May 18, 1977. at the 68th Annual Meeting of the AmericanAssociation for Cancer Research, Denver, Colo.

2There is no unanimity of opinion on the possible roles of viruses in thecausation of human cancer. While most investigators today appear to findthe available data unconvincing as evidence for an important role ofinfectious viruses, Gross (60) believes that viruses may be key factors in theetiology of human cancer.

JUNE 1978 1479

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E. C. Miller

Table 1Chemicals generally recognized as carcinogens in the human

ChemicalIndustrial

exposures2(or/3)-NaphthylamineBenzidine

(4,4'-diaminobiphenyl)4-Aminobiphenyl

and4-nitrobiphenylBis(chloromethyl)etherBis(2-chloroethyl)sulfideVinylchlorideCertain

soots, tars,oilsChromiumcompoundsNickelcompoundsAsbestosAsbestos

plus cigarettesmokingMedical

exposuresA/,/V-Bis(2-chloroethyl)-2-naphthylamine(Chlornapthazine)DiethylstilbestrolSocietalCigarette

smokeBetel

nut and tobacco quidsSites

of tumorformationUrinary

bladderUrinarybladderUrinarybladderLungsRespiratory

tractLivermesenchymeSkin,lungsLungsLungs,

nasalsinusesPleura,peritoneumLungs,

pleura, peritoneumUrinary

bladderVaginaLungs,

urinary tract, pancreasBuccal

mucosaRef.84,

Vol.484,Vol.184,Vols. 1and84,Vol.484,Vol.984,Vol.784,Vol. 3;10384,

Vol.284,Vols. 2and84,Vols. 2and84,Vols. 2and84,

Vol.484,

Vol.61911514111414

Early Studies

The beginnings of our knowledge on chemical carcino-genesis can be traced to two observations in London,England. The first was that of the astute physician John Hill(172) in 1761 on the development of nasal cancer as aconsequence of excessive use of tobacco snuff, and thesecond was that of the perceptive surgeon Percival Pott(164) on the unusually high incidence of cancer of the skinof the scrotum among young men who were chimneysweeps in their childhood. Pott's observation was appar

ently the basis of the first preventive measures againstchemically induced cancers in humans, since, according toClemmesen (33), 3 years later the Danish chimney sweeper's guild urged its members to take daily baths. About

100 years later Butlin (27), another English physician, concluded that the low incidence of scrotal cancer among thechimney sweeps in northern Europe as compared to thehigh incidence among English chimney sweeps was relatedto the better personal hygiene and protective clothing ofthe former group.

The development of skin cancer in certain workers wasshown by von Volkmann in Germany in 1875 and by Bell inScotland in 1876 to be associated with contact of the skinwith tar and paraffin oils that, as we now know, containedpolycyclic aromatic hydrocarbons (reviewed in Ref. 64).The latter observations led in 1907 to the inclusion of suchskin cancers in the third schedule of the British Workmen's

Compensation Act (75). These observations were followedin the latter part of the 19th century by Rehn's acute

observations (173) on the development of cancer of theurinary bladder in three workers in a so-called "aniline" dye

factory in Germany and by the subsequent observations inmany countries on the association between human bladdercancer and occupations that resulted in relatively grossexposures to 2 (or /3)-naphthylamine, benzidine (4,4'-dia-minobiphenyl), or 4-aminobiphenyl (Ref. 29, pp. 40-45).

These observations on higher incidences of specific cancers in individuals with particular chemical exposures

stimulated attempts to induce tumors in experimental animals by application of the implicated chemicals and relatedsubstances. Fischer (52) met with some success in 1906,when he found that the application of the azo dye scarletred (1-[4-(o-tolylazo)-o-tolylazo]-2-naphthol) induced a pro-liferative lesion of the skin in rabbits. However, theselesions did not progress to frank neoplasia and regressedafter the applications of scarlet red were stopped. A numberof investigators sought to demonstrate the carcinogenicactivity of soots and tars in experimental animals. Successwas achieved in 1915 by Yamagiwa and Ichikawa in Japanwho induced carcinomas on the ears of rabbits by repeatedtopical applications of coal tar for long periods (see Ref.64). Tsutsui in 1918 then showed that tars are also carcinogenic for mouse skin, and in 1922 Passey induced cancer inmouse skin by application of ether extracts of tars (64).

The induction of skin cancer with tars and extractsthereof led to searches for the active agents. Chemicalstudies by Bloch and Dreifuss and their collaborators inGermany suggested that polycyclic aromatic hydrocarbonswere the active materials (64). More conclusive evidencewas available when Kennaway (96) in 1925 produced carcinogenic tars by pyrolysis in a hydrogen atmosphere ofseveral organic materials, including acetylene. Hieger's

work (77) revealed that the fluorescence spectra of productsfrom the tars and of synthetic benz(a)anthracene derivativeswere similar. This observation led to the demonstration byKennaway and Hieger (97) in 1930 of 1,2,5,6-dibenzanthra-cene3 or, as it is now known, dibenz(a,h)anthracene, as thefirst synthetic carcinogen (Chart 1). Soon thereafter, acarcinogenic hydrocarbon isolated from coal tar was identified by Cook, Hewett, and Hieger (35) as 3,4-benzpyrene,now called benzo(a)pyrene. Extensive studies, especially byKennaway and his associates in England and by Shear,Fieser, and their associates in this country, soon led to a

3The hydrocarbon was designated as 1,2,7,8-dibenzanthracene in theoriginal paper (97). According to Hartwell (Ref. 69, p. 238) it should havebeen called 1,2,5,6-dibenzanthracene.

1480 CANCER RESEARCH VOL. 38



Current Perspectives on Chemical Carcinogenesis

rororc? Â«WDIBENZIo.hlANTHRACENE BENZOfolPYRENE

3-METHYLCHOLANTHRENE 7,12-DIMETHYLBENZIol-

ANTHRACENE

,CHj

2',3-OIMETHYL-4-AMINO- N.N-DIMETHYL-4-AMINO-

AZOBENZENE AZOBENZENE

Chart 1. The structures of the principal chemical carcinogens discoveredprior to 1940.

large literature on the chemical features required for thecarcinogenicity of the polycyclic aromatic hydrocarbons(reviewed in Ref. 29, pp. 137-164; Ref. 40). Of the carcinogenic polycyclic aromatic hydrocarbons studied during thisearly period, benzo(a)pyrene, 3-methylcholanthrene, di-benz(a,tÃ¬)anthracene,and 7,12-dimethylbenz(a)anthracenehave been most widely used in subsequent experimentalstudies.

Following the early work of Fischer, Yoshida (235)showed in 1933 that p.o. administration of a derivative ofscarlet red, o-aminoazotoluene or 2',3-dimethyl-4-amino-

azobenzene, induced liver tumors in rats and mice. Threeyears later Kinosita (102) demonstrated the strong carcinogenicity of an isomer, /N/,/V-dimethyl-4-aminoazobenzene. In1938 Hueper, Wiley, and Wolfe (82) succeeded in theinduction of cancer of the urinary bladder in dogs fed 2-naphthylamine. Thus, by 1940 the epidemiological data onthe carcinogenicity of an aromatic amine and of coal tarsand soots for man had been complemented by definitivedata on the carcinogenicity of pure chemicals contained inthese mixtures for laboratory animals. Furthermore, in 1932Lacassagne (109) made the first observations on the development of mammary cancer in male mice treated withestrone and thus opened the large field of hormone-induced tumors for experimental study.

This limited list of known carcinogenic chemicals expanded markedly in the 1940's (Chart 2). The carcinogenicity of 2-acetylaminofluorene was first observed by Wilson,DeEds, and Cox (228) in 1941. Subsequent studies showedthe versatility of its carcinogenic activity for various tissuesand species (223), and a number of related amides werefound to have similar activity (138, 139, 143). Also in 1941Edwards (43) reported the induction of hepatomas in miceby carbon tetrachloride; a number of halogenated hydrocarbons have since shown similar activity. Urethan (ethylcarbamate) was found by Nettleship and Henshaw (153) toinduce adenomas of the lung in mice, and subsequentstudies demonstrated the versatility of this carcinogen(146). The induction in 1946 of osteosarcoma in rabbits byzinc beryllium silicate and beryllium oxide by Gardner andHeslington (57) was the first experimental demonstration of

2-ACETYLAMINOFLUORENE(N-2-FLUORENYLACETAMIDE)

ETHYL CARBAMATE

CHo-CHo-CI'

CARBONTETRACHLORlOe

B>O

BERYLLIUM OXIDE

CHjCH3-N N-NO

1CH2-CH2-O CHj'

N-METHYL-BIS- DIMETHYLNITROSAMINE(ÃŸ-CHLOROETHYD- AMINE

CHj-Q^-S-CHj-CHj- CH- COOH

NH2

Chart 2. The structures of some chemical carcinogens identified between1940 and 1960.

the carcinogenicity of certain inorganic chemicals (Ref. 29,pp. 113-134). Similarly, the carcinogenicities of thiourea,thioacetamide, and the nitrogen mustards were first observed in this decade (20, 53, 168).

Data reported in the 1950's revealed the carcinogenic

activities of new classes of chemicals: the wide range ofalkylating agents (112); the dialkylnitrosamines (119); ethi-onine (48); and the pyrrolizidine alkaloids (34, 183). Thecarcinogenicity of the pyrrolizidine alkaloids, like that ofestrone, made it evident that chemical carcinogens are notsolely the products of chemists or of high-temperaturecombustions. A number of metabolites of plants and microorganisms are now known to be carcinogenic for experimental animals (Chart 3) (142), and it is probable that manymore naturally occurring carcinogens exist among the vastnumber of uncharacterized nonnutrient metabolites of living cells. Some of these can be expected to contact humantissues, usually in low doses, through food or as productsof the intestinal flora.

The large number and great variety of organic chemicalcarcinogens now known belong to over a dozen differentclasses. A much smaller number of inorganic chemicalcarcinogens have been identified, and many classes ofinorganic chemicals remain to be tested for their carcinogenic potentials (69, 84, 91, 185, 208).

An achievement of great importance was the development of techniques for the malignant transformation ofcells in culture by chemicals. The first reproducible systemfor malignant transformation was reported in 1963 by Ber-wald and Sachs (12), who used hamster embryo cells. Themaintenance and transformation of rodent fibroblast cultures have since been examined in considerable detail.Systems for the transformation of epithelial cells have beenmore difficult to develop. This important area of research,which has provided valuable techniques for elucidation ofthe molecular events that occur during chemical carcino-genesis, has been critically reviewed by Heidelberger (73).

Tumor Induction as a Multistage Phenomenon

Skin. The classical methods for the induction of tumorsby chemicals have usually involved the administration of asingle agent, most often in a repetitive manner, with the

JUNE 1978 1481



E. C. Miller

HOHjC

OH

CYCASIN(CYCAD TREE FERNS)

R'-O CH0-0-C-Rb'

0-CH3

AFLATOXIN B,

(ASPERGILLUS FLAVUS)

PYRROLIZIDINE ALKALOIDS(SENECIO,CROTOLARIA AND

HELIOTROPIUM GENERA)

OCHj

MITOMYCIN C

(STREPTOMYCES CAESPITOSUS)

CI CH3

GRISEOFULVIN

(PENICILLIUM

GRISEOFULVUM)

Chart 3. The structures of some chemical carcinogens that are productsof plants and microorganisms.

end point being the development of benign or malignanttumors or both. However, it has been evident for manyyears that the induction of skin tumors in the mouse andrabbit is a multistage process.

At first shown by Rous and his associates (55, 179),Mottram (150), and Berenblum (9) and further developed byBerenblum and Shubik, Boutwell, and Van Duuren andtheir associates, the administration of a limited dose of achemical carcinogen to mouse or rabbit skin causeschanges in some cells that are imperceptible in the absenceof further treatment and do not by themselves result in tumors (reviewed in Refs. 16, 17, and 213). This initiation canbe effected by a single dose of the carcinogen, such as analkylating agent, polycyclic aromatic hydrocarbon, or ethylcarbamate. Initiation is generally considered to be completed rapidly and to be essentially irreversible. The second stage, promotion, occurs over a period of weeks andmonths and is, at least in its early phases, largely reversible.The classical promoting agent for mouse skin is croton oilfrom the seeds of Croton tiglium; it was first used byBerenblum as a cocarcinogen (9). Structural characterization of the active ingredients of the croton oil as 12,13-diesters of the diterpene alcohol phorbol (Chart 4) wasaccomplished through independent studies in several laboratories, especially those of Hecker and of Van Duuren(see Refs. 71 and 213). Of these diesters tetradecanoylphor-bol acetate is by far the most active; anthralin (1,8-dihy-droxy-9-anthrone), which is much less active as a promoterthan the latter ester, is the most active of the nonphorbolderivatives studied (213).

Chart 5 illustrates some essential features of the mouseskin tumor initiation-promotion system. Administration ofonly a single small dose of an initiator such as 7,12-dimeth-ylbenz(a)anthracene or of only repetitive doses of the promoter tetradecanoylphorbol acetate does not lead to grosstumors (16, 213). However, sequential administration of thehydrocarbon and then repeated doses of the phorbol estergive rise to a large number of papillomas within 3 to 4months and carcinomas in about 1 year. Similar results areobtained even if several months elapse between the application of the initiator and the first dose of promoter (11,215). Important facts are that the sequence of application

Chart 4. The structure of 12-O-tetradecanoylphorbol-13-acetate or phor-bol-12-myristate-13-acetate, a very potent promoter for tumorigenesis inmouse skin. This promoting agent is found in the seeds of Croton tiglium.

-TIME 0

I = INITIATOR .single dose)

P- PROMOTER i many doses)

Chart 5. Diagrammatic representation of the two-stage induction of tumors in the skin of the mouse. Typical initiators are the polycyclic aromatichydrocarbons, ethyl carbamate, and certain alkylating agents. The mostpotent promoting agent is 12-O-tetradecanoylphorbol-13-acetate (Chart 4)Papillomas develop within about 12 to 20 weeks, and carcinomas develop atabout 1 year.

of the initiator and promoter cannot be reversed and thatpromotion requires repeated doses of the promoter.

Liver. About 20 years ago Weiler (219) observed islandsof morphologically normal hepatic cells in livers from ratsgiven N,W-dimethyl-4-aminoazobenzene and then maintained on a dye-free diet for relatively long periods. Asfurther elucidated by Hughes (83), these cells could bedistinguished from the majority of the hepatic cells by theirdecreased ability to adsorb fluorescein-conjugated globulins, but they were often not recognized as different fromother liver cells on staining with hematoxylin and eosin.The studies on such altered, possibly premalignant cells,have been greatly extended by the groups working withFarber, Becker, Friedrich-Freksa, Rabes, and Emmelot (seeRefs. 49, 50, 181, and 182). Farber, Becker, and theircolleagues have designed protocols for the development ofgross nodules of apparently nonneoplastic hepatic cells. Inrats subsequently maintained in the absence of the carcinogen, the cells of many of these hyperplastic nodulesceased to proliferate and were reincorporated into more orless normal hepatic structures (reviewed in Ref. 50).

A two-stage system of hepatic tumor formation was introduced in 1971 by Peraino and his associates (158, 159),who used a limited period of administration of 2-acetylami-nofluorene and subsequent long-term dosing with pheno-





barbital. The result of the combined treatment was a highincidence of relatively highly differentiated hepatocellularcarcinomas, while the limited treatment with 2-acetylami-nofluorene alone induced far fewer hepatic tumors. Long-term administration of phÃ©nobarbital to rats induced notumors (158, 159) or a low incidence after a long latentperiod (178).

Modifications of these systems are now being examinedby many investigators. In preliminary studies Pitot (162)obtained hepatocellular carcinomas 1 year after administration of a single dose of diethylnitrosamine, 5 mg/kg, topartially hepatectomized rats that were, after 2 months, fedphÃ©nobarbitalcontinuously in the diet. The livers of theserats also contained large numbers of "foci" of hepatic

parenchymal cells that were distinguishable from normalcells by histochemical, but not morphological, techniquesat the light microscope level. These groups of cells, whichcontained as few as 500 cells, were recognized by theirdeficiencies in histochemically detectable glucose-6-phos-phatase or canalicular ATPase or by increases in y-gluta-myltranspeptidase. The cells in these foci differed fromnormal parenchymal cells in 1, 2, or all 3 of these enzymaticactivities (Chart 6), and the individuality of such cells willprobably become even more evident as further markers arestudied. Far fewer of these groups of altered cells wereobserved in livers from rats given only the low dose ofdiethylnitrosamine, and they were rarely seen in livers fromrats given only the phÃ©nobarbital. The broad diversity ofphenotype from the earliest recognizable altered cells tothe primary tumors and the elucidation of the precursor-product relationships, if any, between these two classes ofcells will be exciting areas of research in the new few years.

Other Tissues. Although the models have been muchless well developed than for mouse skin tumors or rat livertumors, evidence has also been presented for multistageinduction of tumors of the mammary gland (4), thyroid (67),lung (3), urinary bladder (25, 76), and certain other tissues.Some of the problems in the interpretation of these datahave been discussed by Berenblum (10).

The two stages of initiation and promotion have alsobeen demonstrated in mouse fibroblast cultures. Thus, asshown by Lasne and his associates (110) and by Mondai efal. (148), a single short treatment with less than 1 ^g of apolycyclic hydrocarbon did not give rise to clones of transformed cells. However, the subsequent exposure of thecells to 12-tetradecanoylphorbol-13-acetate gave rise tolarge numbers of clones of transformed cells. The numberof transformed cells was dependent both on the amountand structure of the initiating agent and on the structure ofthe promoting agent.

Electrophilicity as a Common Property of Ultimate Carcinogens

Several observations early suggested that the metabolismof chemical carcinogens might be a key factor in theircarcinogenic activities. Thus, as the number and variety ofchemical carcinogens increased, it became more and moreevident that these chemicals lacked a common structuralfeature (Charts 1 to 3). Furthermore, some carcinogens,especially the aromatic amine derivatives, produced tumors

at distant sites such as the liver and urinary bladder regardless of the route of administration. An early clue to thepossible metabolic activation of a carcinogen was ourfinding in 1947 of the covalent binding of a metabolite of/V,/V-dimethyl-4-aminoazobenzene to the hepatic proteinsof rats fed this dye (131). Similar observations on theformation in target tissues of protein-bound derivatives ofthe polycyclic aromatic hydrocarbons and 2-acetylamino-fluorene soon followed in our laboratory and those ofHeidelberger and the Weisburgers (reviewed in Refs. 72,74, 132, 133, and 223).

Other studies, especially in the laboratories of Heidelberger (74, 206), Ketterer (98), and Sorof (192) and theirassociates, showed marked specificities of certain proteinsof the target tissues for binding to these carcinogens. Thereasons for these specificities and the roles of these protein-bound carcinogen derivatives in carcinogenesis havenot been elucidated, but Mainigi and Sorof (121) haverecently reported data consistent with their suggestion thatthe principal hepatic azo protein is a vehicle for the intra-cellular transport of an ultimate carcinogenic derivative of3'-methyl-/V,/\/-dimethyl-4-aminoazobenzene.

The recognition of the central roles of DNA's as storehouses of genetic information and of RNA's in the transla

tion of the genetic information for the synthesis of cellularproteins provided new perspectives on the possible roles ofnucleic acid-carcinogen adducts in carcinogenesis andprovided an impetus to search for such derivatives. Wheelerand Skipper (226) reported in 1957 that 14Cfrom [methyl-14C]bis(2-chloroethyl)methylamine was incorporated into

the purine fractions from the RNA and DMA of certainmouse tissues. Subsequent studies by Farber and Mageeand their colleagues (51, 118, 123), by Brookes and Lawley(24), by Heidelberger (72), and by Stekol ef al. (195) soondemonstrated the incorporation of 14C from 14C-labeledethionine, 2-acetylaminofluorene, dimethylnitrosamine,and polycyclic hydrocarbons into the DNA and RNA of thetarget tissues. Since that time the administration of allcarcinogens that have been adequately studied has yieldedDNA-, RNA-, and protein-bound derivatives in the targettissues (74, 133). Correlations between the levels of thesenucleic acid- and protein-bound derivatives and the likelihood of tumor development were obtained in many, but notall, cases. Taken as a whole, the data indicated that macro-molecular-bound forms of the carcinogens were a necessary, but not sufficient, correlate for the induction of tumors by chemical carcinogens.

Gradually, these and other studies led to the generalization that the great majority of chemical carcinogens wereactive only after metabolism to ultimate carcinogens (;'.e.,

the derivatives that actually initiate the neoplastic event).The known exceptions are the carcinogens that are alkylat-ing or acylating agents per se. Further, the data thenavailable suggested to us that the ultimate forms of chemical carcinogens might all be strong electrophilic reactants(Chart 7) (141). This conclusion still appears to be valid,although a few carcinogens, such as Adriamycin (122), maybe active through tight noncovalent binding rather than asa result of covalent reaction with a macromolecule. Thus,the known ultimate carcinogens contain relatively electron-deficient atoms that seek to react with nucleophilic sites,

JUNE 1978 1483



E. C. Miller

Chart 6. Biochemical phenotypes of foci of altered parenchymal cells in asection of liver from a rat given 1 p.o. dose (5 mg/kg) of diethylnitrosamine24 hr after a partial hepatectomy and, starting 2 months later, 0.05%phÃ©nobarbitalin the diet for 6 months. The enzyme activities were determined on serial sections. O, glucose-6-phosphatase-deficient areas, ,canalicular ATPase-deficient areas; â€¢y-glutamyltranspeptidase-posi-tive areas. (This chart was kindly provided by Dr. H. C. Pitot of the McArdleLaboratory.)

H H

:NU =9 _R-O-tf-0 , R-SOR'

Chart 7. Examples of strong electrophihc reactants (positive ions oruncharged molecules with electron-deficient atoms) and their reactions withnucleophiles (:NU) through sharing of electron pairs of electron-rich atoms.

i.e., atoms that have easily shared electrons. These nucleophilic sites are relatively abundant in DNA's, RNA's, and

proteins and include certain oxygen and nitrogen atoms inthe nucleic acids and nitrogen, sulfur, and oxygen atoms inproteins (Chart 8). Because some, and probably many,precarcinogens are metabolized to more than one ultimatecarcinogen and because there are multiple nucleophilicsites in each macromolecule, multiple DNA-, RNA-, andprotein-bound derivatives of each carcinogen are possible

and are frequently observed. Accordingly, basic problemsof great importance today are the elucidation for eachcarcinogen of those informational macromolecule-bound

products that are important in carcinogenesis and the

identification of the specific role of each adduct in thecarcinogenic process.

Since carcinogenic chemicals are promoters as well asinitiators, it is possible that carcinogen-macromolecule

interactions are of fundamental importance in both phasesof the overall process. Furthermore, since there are initiators with little or no promoting activity (e.g., ethyl carba-

mate in mouse skin) (180, 213) and promoters with little orno initiating activity (e.g., phorbol esters) (71,150, 213), theultimate initiating and ultimate promoting agents from agiven precarcinogen may be either identical or different.Furthermore, carcinogenesis by a chemical that has bothinitiating and promoting activities may require interactionwith more than one kind of macromolecule, e.g., with bothDNA and specific proteins.

Examples of the Metabolic Activation and Reactivity ofChemical Carcinogens

Potential Donors of Simple Alkyl Groups. This groupincludes especially the dialkylnitrosamines, dialkylhydra-

zines, aryldialkyltriazenes, and alkylnitrosamides. The firstthree classes of these versatile carcinogens are metabol-ically dealkylated by the mixed-function oxidases in the

endoplasmic reticulum, and these monoalkyl derivativesspontaneously decompose to the corresponding monoal-kyldiazonium ions (112, 119) (Chart 9). The W-nitrosimidesand A/-nitrosamides do not require enzymatic activation,

since their reaction with water and other cellular nucleophiles results in the formation of the same alkylating inter-

CELLULAR NUCLEOPHILEla+--- â€”ti

DISPLACEDELECTROPHILE

ELECTROPHILIC REACTANT^ (x+-- --y") >bx

ELECTROPHILIC LEAVINGATOM NUCLEOPHILE

NONEORH*:S

MET-SHCYS*

(HIS1N-I.N-3)/GIN-3.N-7.N2)'^AtN-l

N-3N-7)jC(N-3)YH

TYRIC-3)'G(C-B)*C-OHTYR6<Â°''l3=-OH

DMA-C*>R-SCÃ•.R-COÂ¿,HSOÂ¿H

PCÃ»CI"21^N~,-0"

fromstrained

ringsR-COj

, HS04

Chart 8. In vivo macromolecular nucleophilic targets of chemical carcinogens that have been identified up to the present.

DIMETHYLNITROSAMINE N-METHYL-N-NITROSOUREA

N-NO

,cH2N 0

itHop, non-enzymatic

r *; -iCH3|N=NfOH

J, DNA, RNA, PROTEIN

CHj-ONA, CHj-RNA WITH cf-CHj-G, 7-CH3-G, 3-CHj-A, ETC.CH3-PROTEIN WITH I- and Ã®-CHj-HISTIDINE.S-CHj-CYSTEINE, ETC.

Chart 9. The in vivo conversion of dimethylnitrosamine and of N-methyl-N-nitrosourea to a reactive electrophile and its reaction with cellular macro-molecules.





mediates. Administration of these carcinogens results inthe alkylation, to various degrees, of a wide variety ofnucleophilic sites in the target cells.

2-Acetylaminofluorene. 2-Acetylaminofluorene is a morecomplex carcinogen for which the metabolic activation hasbeen worked out in some detail for one target tissue (theliver) (Chart 10). Studies with Cramer in our laboratory in1960 showed that rats fed 2-acetylaminofluorene convertedit to a new metabolite, A/-hydroxy-2-acetylaminofluorene,which is a stronger carcinogen than is the parent compound and which is also active in a wider range of tissuesand species (36, 136, 137, 140). Although administration ofboth 2-acetylaminofluorene and its /V-hydroxy derivativeyielded nucleic acid- and protein-bound derivatives, especially in the rat liver (107, 133, 223), these carcinogens arenot reactive in vitro and further metabolism seemed necessary. Studies by King and Phillips (101) and by DeBaun inour laboratory (37) showed the presence of soluble sulfo-transferase activity for N-hydroxy-2-acetylaminofluorene inrat liver; the product of this reaction, /V-sulfonoxy-2-acetyl-aminofluorene, appears to be a major ultimate carcinogenicmetabolite in rat liver. Thus, hepatic sulfotransferase activity under various conditions correlates with susceptibility tohepatic tumor formation, the sulfuric acid ester is a verystrong electrophile, and the hepatotoxicity and hepatocar-cinogenicity of W-hydroxy-2-acetylaminofluorene were decreased on reduction of the amount of available activesulfate or 3'-phosphoadenosine 5'-phosphosulfate in vivo(37, 38, 224). The biological activity of the sulfuric acidester was also evident from its high mutagenic activity in aDMA-transforming system (120).

In spite of the apparent major importance of the sulfuricacid ester for liver tumor formation in the rat, it should benoted that three other enzymatic pathways for conversionof /V-hydroxy-2-acetylaminofluorene to electrophilic reac-tants have also been observed in rat liver (Chart 11). Asshown by Bartsch and Hecker (7), W-hydroxy-2-acetylami-nofluorene undergoes a peroxidase-catalyzed one-electronoxidation to yield a free nitroxide radical, and two of theseradicals can dismutate to yield the electrophiles /V-acetoxy-2-acetylaminofluorene and 2-nitrosofluorene. An electronspin resonance signal that is consistent with the formationof this free radical has been observed by Stier ef a/. (196) onincubation of 2-aminofluorene with rabbit liver microsomes.Further, Bartsch in our laboratory (6) showed that rat liver

COCH, rot liver E.R..,+NADPH+ OZ

/COCHj|XOH

2-ACETYLAMINOFLUORENE(AAF)

AAF- RESIDUES COVALENTLYBOUND TO HEPATICINFORMATIONAL MACRO-MOLECULES

N-HYDROXY-AAF

rot livercy totol

+ PAPS

rot liver DNA,RNA, protein

,COCH3

So-so,AAF-N-SULFATE

PAPS â€¢3-photphoadtnotlne -5- photphoiulfati ("active Â«ulfotÂ«")

Chart 10. The major pathway for the metabolic activation of 2-acetylaminofluorene for carcinogenesis in rat liver. E.R., endoplasmic reticulum.

DISMUTATION*

PEROXIDASE

-C-CH

N-ACETOXY-AAF

2-NITROSOFLUORENE

H2N-ACETOXY-AF

GLUCURONYLTRANSFERASE

+ UDPGA

0-GLUCURONIDE

Chart 11. Pathways, in addition to the formation of the sulfuric acid ester,for the metabolism of/V-hydroxy-2-acetylaminofluorene to electrophilic reac-tants in rat liver. N-ACETOXY-AAF, N-acetoxy-2-acetylaminofluorene; N-ACETOXY-AF, N-acetoxy-2-aminofluorene; UDPGA, uridine diphospho-glucuronic acid.

cytosol forms the very potent electrophile /V-acetoxy-2-ami-nofluorene by enzymatic transfer of the acetyl group fromthe nitrogen atom of /V-hydroxy-2-acetylaminofluorene tothe oxygen atom of the hydroxylamine. Finally, N-hydroxy-2-acetylaminofluorene is converted by hepatic microsomesto the weakly electrophilic O-glucuronide (86, 130). Theseenzymatic reactions are also candidate systems for theformation of ultimate carcinogenic metabolites in extrahe-patic target tissues where sulfotransferase activity for N-hydroxy-2-acetylaminofluorene has not been detected (37,87, 100). The acetyltransferase may be of special importance in view of the wide range of tissues in which thisactivity occurs and the very high reactivity of the product,W-acetoxy-2-aminofluorene (6, 99, 100).

Both acetylated and nonacetylated aminofluorene ad-ducts have been isolated from the livers of rats treated with/V-hydroxy-2-acetylaminofluorene (5, 37, 107, 225) (Chart12). On the basis of the products formed in in vitro reactions, the acetylated adducts must be formed primarily fromesters of /V-hydroxy-2-acetylaminofluorene, while the nonacetylated adducts are presumably derived either fromesters of A/-hydroxy-2-aminofluorene or from reaction of theglucuronide of /V-hydroxy-2-acetylaminofluorene, whichyields a mixture of acetylated and nonacetylated adducts(130). While the methionine adducts (as evidenced by theamounts of o-methylmercapto-2-acetylaminofluorene ando-methylmercapto-2-aminofluorene isolated after degradation of the liver proteins) comprise only about 10% of theprotein-bound fluorene derivatives (5, 37), the guanineadducts that have been identified appear to account for themajor share of the nucleic acid adducts formed in rat liverin vivo. The major adducts are those in which the substitution occurs at C-8 of guanine (107), and the latter adductsare much more readily removed in vivo from the DMA of ratliver than is the minor adduci in which the substitutionoccurs on the 2-amino group of guanine (106, 225).

JUNE 1978 1485



E. C. Miller

NUCLEIC ACID ADOUCIS

PROTEIN ADDUCTS

PROTEIN

Chart 12. Nucleic acid- and protein-bound adducts that have been identified in the livers of rats treated with 2-acetylaminofluorene or N-hydroxy-2-acetylaminofluorene.

An important question to be answered in considering thesubstitution of macromolecules by chemicals in relation tocarcinogenesis is how the alterations affect the activities ofthe molecules. Fuchs ef al. (56) and Weinstein and Grun-

berger (220) have both considered this problem for DNAsubstituted with 2-acetylaminofluorene residues at C-8 of

guanine, and they have interpreted their structural findingswith similar models in which the guanine of the adduci isrotated out of the double helix and the fluorene moiety isinserted into the helix. These models are consistent withbase-pair substitution, frame shift, and deletion mutations,all of which have been observed with derivatives of 2-

acetylaminofluorene (46, 120, 127). In addition, this substitution causes premature termination of transcription (144)and interferes with codon recognition of tRNA's (220).

Other Aromatic Amines and Nitro Compounds. The activities of all carcinogenic aromatic amines, amides, andnitro compounds appear to depend on their conversion toN-hydroxy derivatives in vivo (30, 140, 141). The ultimate

carcinogenic metabolites have not been elucidated in mostcases, and the activation reactions may differ with the arylsubstituents, tissues, and species. For instance, /V-methyl-4-aminoazobenzene is activated similarly to 2-acetylaminofluorene by /V-hydroxylation and sulfonation of the /V-hy-

droxy derivative, and the major nucleic acid adducts involvesubstitution of C-8 of guanine residues by the nitrogenatom of /V-methyl-4-aminoazobenzene (92, 93, 114). However, while the oxidation of 2-acetylaminofluorene is catalyzed by a cytochrome P-450 system, the /V-oxidation of the

dye is catalyzed by a flavoprotein that does not requirecytochrome P-450, and the data indicate that different

hepatic sulfotransferases may act on the two substrates.The carcinogen 4-nitroquinoline 1-oxide is reduced to 4-hydroxyaminoquinoline 1-oxide, and Tada and Tada (204)

have shown that this hydroxylamine can be esterified byseryl-tRNA, The resulting seryl ester, which has been sug

gested as an ultimate carcinogenic metabolite, reacts primarily with guanine and, to a lesser extent, with adenineresidues.

Recent studies have provided evidence that the nitrenium

ions formed on protonation of N-hydroxy-2-naphthylamineand N-hydroxy-4-aminobiphenyl may be ultimate carcino

gens for the induction of urinary bladder tumors in the dogand human (Chart 13). Thus, Kadlubar in our laboratory (94)showed that these hydroxylamines are N-glucuronidated by

the hepatic endoplasmic reticulum from these species, andRadomski ef al. (170) have recently characterized the N-glucuronide of /V-hydroxy-4-aminobiphenyl as a urinarymetabolite of 4-aminobiphenyl in the dog. Furthermore, the

urines of many dogs and humans are sufficiently acidic tohydrolyze the /V-glucuronides and to protonate the resulting

hydroxylamines for reaction with DNA (94). These conclusions are consistent with the earlier report by Radomskiand Brill (169) on the quantitative correlation between thecarcinogenicities of 1- and 2-naphthylamine and 4-amino

biphenyl in the dog urinary bladder with the level of excretion of the corresponding A/-hydroxylamines (plus the nitro-

scarenes). Furthermore, bladder carcinomas were inducedin dogs by the instillation of N-hydroxy-2-naphthylaminebut not 2-naphthylamine.

Polycyclic Aromatic Hydrocarbons. Studies on the metabolism of the polycyclic aromatic hydrocarbons were firstreported in the late 1930's, and by 1950 Berenblum,

Schoental, Weigert, Mottram, Dobriner, and their associates had observed phenolic and quinone derivatives ofseveral polycyclic hydrocarbons in tissue preparations orexcreta of animals treated with these compounds (reviewedin Ref. 29, Chap. 7). The knowledge of the sites and extentsof this metabolism was greatly extended during the nexttwo decades, especially by reports from the laboratories ofBoyland and Sims and of Heidelberger (reviewed in Refs.40, 74, and 187).

* NADPH endoplasmic+ 02 reticulum

V endoplasmicA M--OHreticulumAr

, H ,UDPGA

|1

1,.

V0"PH<7ArN,H<Â¡

pH>7\^Â¿'Ar-IS1A

>H*HÂ®,-H,01

1 '

Â¡METABOLIC

1 ACTIVATIONVREACTIVE

ELECTROPHILES

(ESTERS')( FREE RADICALS ~>)COW

OHLIVERti/HVN^ArH

OHTRANSPORTH/â€”\

N'Â°HHoTTHArÃ•TÃ•H

URINEVH

^H-O /â€¢VHÂ¿X^.

Ar MÂ®URINARYlH BLADDER

Â¡EPITHELIUMCOVALENT

BINDING TO> NUCLEOPHILIC SITES IN

CRITICAL MACROMOLECULES

TUMOR FORMATION

Chart 13. Formation and transport of possible proximate and ultimatecarcinogenic metabolites of arylamines for the induction of urinary bladdercancer. , possible transport or reaction. Ar, aryl substituent; UDPGA,uridine diphosphoglucuronic acid (from Ref. 94).





As early as 1950 Boyland (19) suggested that the series ofmetabolic phenols and dihydrodiols might be secondaryproducts of metabolically formed epoxides and that theseepoxides might be intermediates in tumor induction. At thattime the K-regions of the hydrocarbons (i.e., the phenan-threne-like double bonds) had been singled out by thePullmans (166) from calculations of electron densities aslikely critical sites for the interactions of the hydrocarbonswith tissue constituents, and the K-region epoxides weretherefore the first epoxides to be examined for carcinogenicactivity. The low carcinogenicities of the K-region epoxidesof benz(a)anthracene or related hydrocarbons on s.c. injection or topical application to rats or mice in our laboratoryand those of Boyland, Sims, and Van Duuren were disappointing (21, 134, 186, 214). However, studies in Heidelber-ger's laboratory in 1971 and 1972 showed that usually, but

not always, the K-region epoxides were more active thanwere the parent hydrocarbons for the transformation ofmouse fibroblasts in culture (62, 80).

In 1971 Selkirk ef al. (184) and Grover ef al. (61) showedthe formation by liver microsomes of unidentified epoxidesfrom benz(a)anthracene and dibenz(a,/?)anthracene. Workfrom these laboratories also showed the electrophilic reactivity of the K-region epoxides (108). Knowledge of themechanisms of metabolic activation of the polycyclichydrocarbons and of the possible roles of the metabolicproducts in carcinogenesis has since developed rapidly,especially in the laboratories of Brookes, Conney, Gelboin,Harvey, Jerina, Sims and Grover, and Weinstein. In 1974Sims and his colleagues (188) expanded on an observationof Borgen ef al. (14) that indicated that the critical metabolism of the polycyclic aromatic hydrocarbons might occurat sites other than the K-regions; much evidence for thisconcept has been presented since.

In the past few years particular attention has been focused on the nature of the ultimate carcinogenic metabolites of benzo(a)pyrene and on the identities of its nucleicacid-bound derivatives (Chart 14). Elegant studies from theabove laboratories now implicate 7/3,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene as a major ultimate electrophilic, mutagenic, and carcinogenic metaboliteof benzo(a)pyrene (81, 90, 95, 113, 221). As reported from

10

DNA

ULTIMATECARCINOGEN

Chart 14. The major route for the metabolic activation of benzo(a)pyreneand the major adduci formed on reaction of the diol-epoxide with nucleicacids. E.R., endoplasmic reticulum.

three laboratories, the major reaction products of this diol-epoxide and its 9/3, 10/3 isomer with polyguanylic acid ornucleic acids involve the 2-amino group of guanine residues and C-10 of the epoxide (105, 156, 221). The syntheticadducts are chromatographically identical with productsobtained on degradation of the nucleic acids from bronchialexpiants that had been incubated with [3H]benzo(a)pyrene(221). The diol-epoxide also reacts in vitro, but to a muchsmaller extent, with the cytosine and adenine residues ofpolynucleotides (129, 222).

The analogous diol-epoxide derivative of benz(a)-anthracene was suggested as an ultimate carcinogenicmetabolite of that hydrocarbon by Swaisland ef al. (199).However, data from the laboratories of Conney and Jerinanow strongly indicate that the bay region or 1,2-position ofthe angular ring of benz(a)anthracene may be analogous tothe 9,10-position of benzo(a)pyrene in the formation of anultimate carcinogenic diol-epoxide. Thus, the 3,4-dihydro-diol of benz(a)anthracene is considerably more carcinogenic for mouse skin than is benz(a)anthracene or any ofthe other vicinal dihydrodiols (234), and the 1,2-epoxideformed from the 3,4-dihydrodiol of benz(a(anthracene is amuch more potent mutagen without tissue activation thanare the two isomerie diol-epoxides with the substituents inthe 8, 9, 10, and 11 positions (233). The angular ring hasalso been implicated as a critical site for the metabolicactivation of 7-methylbenz(a(anthracene (207) and of 7,12-dimethylbenz(a)anthracene (149).

Unfortunately, it is impossible to give adequate recognition in this brief review to all of the extensive work on themechanisms by which the polycyclic aromatic hydrocarbons exert their carcinogenic activities. These studies havegiven important new insights into hydrocarbon carcinogenesis as well as further demonstrating the complexities ofthe metabolic activations. Nevertheless, the knowledge inthis area is far from complete. Just as studies on thealkylation of DNA by simple alkylating agents have shownthat the major adducts are not necessarily the ones that arethe most important in carcinogenesis (112, 189, 190), thehydrocarbon-nucleic acid derivatives that have been identified to date may or may not be those adducts most criticalfor the initiation of carcinogenesis. Furthermore, since thepolycyclic hydrocarbons are promoting agents as well asinitiators, attention should also be given to the possiblepromoting activities of the metabolites. Some of the metabolic phenols may be candidates for this role in view of thepromoting activities of a number of phenols (213).

Aflatoxin B,. Several observations led to the suggestionthat aflatoxin B, 2,3-oxide was the most likely ultimatecarcinogenic and reactive metabolite of aflatoxin B,. Theseincluded the requirement of the 2,3-double bond for strongcarcinogenic activity (232) and the conversion by a mixed-function oxidase system of aflatoxin B, (but not its 2,3-dihydro derivative) to a toxic, mutagenic, and nucleic acid-binding derivative (58, 127). More conclusive was therelease by acid hydrolysis of 2,3-dihydro-2,3-dihydroxyafla-toxin B, from nucleic acids isolated from the livers ofaflatoxin B,-treated rats or from incubations of aflatoxin B,and nucleic acids with fortified liver microsomes (201, 202).Finally, the strong electrophile aflatoxin B, 2,3-dichloride,synthesized as an analog of the 2,3-oxide which has thus

JUNE 1978 1487



E. C. Miller

far eluded isolation from chemical or metabolic reactions,was a strong carcinogen at sites of application (e.g.,mouse skin and rat s.c. tissue) and a powerful mutagen(203).

In the past year an acid degradation product of the majoradducts formed on reaction of the epoxide with nucleicacids in microsomal systems in vitro has been independently characterized as 2,3-dihydro-2-(guan-7-yl)-3-hydroxy-aflatoxin B, in our laboratory (115), by Essigmann ef al.(45), and by Martin and Garner (124) (Chart 15). In addition,our data (115) and those of Essigmann ef al. (45) haveidentified the same adduci as a major degradation productof the nucleic acids from the livers of rats treated withaflatoxin B,. While the overall yield of nucleic acid-boundaflatoxin derivatives appears to correlate with the likelihoodof tumor formation (200), there are as yet no data thatspecifically associate the aflatoxin B,-guan-7-yl nucleic acidadducts with carcinogenesis.

Possible Molecular Mechanisms of Chemical Carcinogenesis

The above examples, while far from exhausting the literature on metabolic activation, are sufficient to emphasizethat metabolic activation is an essential step in the induction of neoplasia by most chemical carcinogens. The elec-trophilic ultimate carcinogens can react, probably more orless indiscriminately, with a number of nucleophilic sites inDNA's, RNA's, and proteins. Thus, the strong electrophilic

nature of ultimate carcinogens is consistent with bothgenetic and epigenetic mechanisms of carcinogenesis orwith mechanisms that include both genetic and epigenetic

o rot liverb. liver microsomes

+ NADPHÂ»02

AFLATOXIN B, AFB,-2,3-OXÅ’

ft\DECOMR\PROOUCTS|OFI

[ ]= presumptive structures

Chart 15. The metabolic epoxidation of aflatoxin B, (AFBÂ¡),the majorreaction product of the epoxide with nucleic acids, and the degradation ofthe nucleic acid adducts to yield the major product 2-(guan-7-yl)-3-hydroxy-aflatoxin B, (///). The routes of formation of several other intermediates,especially 2,3-dihydro-2,3-dihydroxyaflatoxin B, (//), on degradation of thenucleic acid adducts are also shown. DECOMP., decomposition (from Ref.115).

events. These mechanisms may or may not involve theexpression of oncogenic viral information (see, e.g., Refs.154, 165, 171, and 175).

Epigenetic Mechanisms. A fundamental basis for proposed epigenetic origins of cancer is the development ofcomplex organisms from single fertilized ova. During earlylife each multicellular organism has many kinds of committed cells that divide repeatedly to give rise to additionalcells with the same commitments. If, as is generally accepted, these normal differentiations are the consequenceof epigenetic phenomena, similar epigenetic modificationsof cellular transcription or translation or both may also beinvolved in the conversion of apparently normal cells totumor cells with relatively stable phenotypes (63). Further,the now classic studies of Jacob and Monod (89) on thecircuits by which genetic expression in bacteria can bemore or less permanently altered, as well as the exquisitecontrols for the expression or repression of information inbacterial genomes (176), provide models for the inductionof tumors by chemicals through proliferation of cell lineswith altered transcriptional controls (163).

Data from a variety of experiments indicate that malignantcells and nonmalignant cells may possess the same genomes, although detailed analyses at the molecular levelhave not yet been feasible. Gurdon (63) and McKinnell ef al.(128) transplanted nuclei from frog renal carcinomas intoenucleated fertilized frog eggs with the subsequent development of apparently normal swimming tadpoles. Thesestudies appeared to show that the nuclei from the tumorsretained in expressible form at least the major share of theinformation that was present in the fertilized ova. Thepotential for the differentiation of malignant cells to non-malignant cells was demonstrated by Pierce and his associates (161) for several types of tumors, including clonedteratocarcinoma cells and stem cells from a transplantablesquamous cell carcinoma. Braun (22) has reported similardifferentiation of plant teratomas. Thus, cultures of terato-mas could be grafted onto a plant where, under someconditions, the progeny of the teratoma cells gave rise tomorphologically normal stems, leaves, and flowers, although the inherent neoplastic potential of the cells wasagain evident on growth in culture (23). On the other handmeiosis apparently caused the loss of the plasmid that isessential for the malignant phenotype, and cells that developed from the seeds grew as normal cells in culture (211).Illmensee and Mintz (85, 145) have recently obtained chi-meric mice by implantation of single cells from embryoidbodies of mouse teratocarcinoma cells into mouse blÃ¡stulas. These chimeric mice contained a wide variety of apparently normal somatic tissues that developed from progenyof the teratocarcinoma cells; in at least one case thegenotype of the teratocarcinoma cell was transferred to thesecond generation through the sperm. Further studies onthe significance of these results in relation to the mechanisms involved in carcinogenesis are awaited with greatinterest.

Genetic Mechanisms. In contrast to the epigenetic hypotheses, which have as their fundamental premise that thegenomic information of tumor cells need not be alteredfrom that of normal cells of the same organism, the geneticmechanisms assume that the change from normal to tumor





cell is dependent on genomic alterations. This latter pointof view receives primary support from the fact that thepotential of a cell is determined by the information coded inthe genome and from the abilities of all three types ofcarcinogenic agents, viruses, radiations, and chemicals, toalter cellular genomic content. Such genomic changes incells would be expected to occur most frequently as a resultof direct modification of DMA by the carcinogen. However,modification of an RNA that was transcribed and integratedinto DMA or changes in the structure of a DMA polymerasethat resulted in a more error-prone enzyme could also leadto altered cellular DNA's.

Auerbach, Demerec, and others were attracted in the1940's to the idea that carcinogenesis might involve muta-

genie events and sought to correlate the carcinogenic andmutagenic activities of chemicals. Their data showed noevident relationship (reviewed in Ref. 26). However, thesituation changed markedly as the metabolism of chemicalcarcinogens and the chemical nature of their active formsbecame better understood. As we noted when we reviewedthis subject in 1971 (135), a qualitative correlation betweenmutagenicity and carcinogenicity was apparent when ultimate carcinogenic forms were assayed in a nonmetaboliz-

ing system (transforming DNA) or when nonultimate formswere assayed in certain cellular systems (e.g., yeast, Dro-sophila) that possessed capacity for metabolism of foreignchemicals. This correlation has become better with thesupplementation of bacterial mutagenicity systems withliver microsomes for the metabolic activation of carcinogens (126, 127, 167, 197, 198). This correlation betweenmutagenic and carcinogenic activities is a formal one andis based on two facts: (a) that the ultimate forms of most, ifnot all, chemical carcinogens are strong electrophilic reac-tants; and (b) that, with the exception of the numericallyminor groups of the base analog mutagens and the simpleframe-shift mutagens that do not bind covalently, the ulti

mate forms of mutagenic chemicals are also strong electrophilic reactants. However, since these strong electrophilicreactants also attack RNA's and proteins, this correlation

cannot be used alone to show that carcinogenesis involvesmutagenic events.

Quite compelling evidence that tumor development maydepend on an alteration of genomic information is availablefor UV-induced carcinogenesis. Thus, xeroderma pigmen-

tosum patients are very susceptible to the development ofskin cancer as a consequence of exposure to sunlight. Asshown by Cleaver, Bootsma, and others, the cells fromthese patients have a greatly impaired capacity for theerror-free repair of DNA that contains UV-induced or certain

chemically induced lesions (reviewed in Ref. 32). Similarly,as reported by Hart and Setlow (68), exposure of cells fromPoecilia formosa to UV/n vitro and subsequent inoculationof the cells into new hosts gave rise to a high incidence ofthyroid tumors. The cells of this species contain a photo-

reactivating enzyme that cleaves pyrimidine dimers, and theexposure of the irradiated cells to visible light after the UVradiation largely prevented the development of tumors onimplantation of the cells.

Support for genetic mechanisms of transformation in cellculture is provided by the formation of stable, but revertible,temperature-sensitive malignant transformants of BHK cells

by treatment with /V-nitrosomethylurea or 4-nitroquinoline1-oxide (15). Furthermore, recent studies by Marquardt ef

al. (122) suggest that Adriamycin, an intercalating drug andframe-shift mutagen, may induce malignant transformation

in cell culture in the absence of detectable covalent interaction with cell constituents.

Modern chromosome banding procedures provide someevidence for associations of specific chromosomal alterations with certain human cancers (152). Likewise, geneticpredispositions for the development of some human cancers can be interpreted as pointing to the involvement ofspecific genetic components (2, 104). However, the interpretation of the latter data in a mechanistic sense is fraughtwith problems, since genetic information also determinesthe metabolism of chemical carcinogens, the synthesis andmetabolism of hormones, and a variety of other factors thatmay affect tumor incidence without being directly involvedin the initiation of cancer cells.

Overall, alteration of cellular DNA is currently viewed bymany and probably most investigators as the most attractivemechanism for the initiation of carcinogenic processes bychemicals. Working on this premise an important questionis the nature of the DNA alterations that may be involved.Some years ago Loveless (116) showed that the extent ofO6-methylation or ethylation of guanine residues in DNA,

reactions that lead to base substitution mutations, correlated much better with the mutagenic activity of an alkylat-ing agent than did the quantitatively more prominent N-7

alkylation of guanine residues. Studies by Lawley andothers (112, 157) suggested a similar correlation of O6-

methylation and ethylation of guanine residues in DNA withthe likelihood of tumor initiation by a series of methylatingand ethylating agents, but exceptions were evident. Thisapproach was refined in 1974 by Goth and Rajewsky (59),who found that the persistence of the O6-ethyl guanine

residues in DNA, in addition to the amount originallyformed, appeared to be a critical factor for the induction oftumors of the nervous system in rats. Further support forthe role of persistent O6-methyl or -ethyl guanine residues

in DNA have been obtained with other carcinogenesissystems, although some apparent exceptions have alsobeen reported (157). Furthermore, as emphasized recentlyby Singer (190), it is too early to conclude that O6-alkyla-

tions of guanine are the most critical reactions, even for theinduction of mutations, since alkylations at this site may beindicators of more critical alkylations at other sites, such asthe oxygen atoms of the pyrimidine bases in the DNA. Tothe extent that chemical carcinogenesis results from attacks on DNA, the great strides that have been made inrecent years in determining the mechanisms by whichspecific types of damage to DNA result in mutations (32,210, 230) will be of key importance in elucidation of themechanisms of carcinogenesis by chemicals.

Promotion of Initiated Cells. New observations are alsoproviding insight into the possible molecular bases ofpromotion. The phorbol esters, which have remarkableactivity in eliciting the development of gross epidermaltumors after application of a small initiating dose of achemical carcinogen to mouse skin, cause a progression ofchanges. Application of these promoters results in increased synthesis of phospholipids, RNA, protein, and DNA

JUNE 1978 1489



E. C. Miller

and an increased mitotic rate (17), but the earliest changesappear to be increases in protease activity (209) and markedincreases in ornithine decarboxylase activity (18). The inhibition of the promotion stage of epidermal carcinogenesisby protease inhibitors suggests that specific proteases maybe of critical importance in promotion (125, 209); increasedlevels of one protease, plasminogen activator, frequentlyaccompany oncogenic transformation in cell culture (174).Similarly, an impressive series of experiments show a closecorrelation between the levels of induced ornithine decarboxylase activity and promoting activity in mouse epidermis. The latter correlation was obtained by comparison ofthe effects of various doses and structures of promoters(155) as well as by comparisons of the extent of inhibitionof tumor promotion and of ornithine decarboxylase induction by a series of retinoid derivatives (217). The inductionsof both protease activity and ornithine decarboxylase activity may have a common focus through their effects on thelevels or intracellular localizations of polyamines or proteins that modulate the expression of the DMAgenome. Theepigenetic concept of promotion is in accord with itsrelatively slow and reversible nature.

Extrapolation of Basic Knowledge of Chemical Carcinogenesis to the Prevention of Human Cancer

Prevention of Initiation. Our still incomplete knowledgeof chemical carcinogenesis is already being applied to theproblem of reducing the future incidences of human cancers. Thus, the generalization that strong electrophilicreactivity is a basic requirement for ultimate chemicalcarcinogens and detailed knowledge of enzyme systemsinvolved in the metabolic activation and deactivation ofmany chemicals are providing useful tools for the recognition of chemicals to be suspected of being potential carcinogens. Reasonable predictions can often be made of theelectrophilic reactivity of a chemical or its possible metabolites from an inspection of its structure. Furthermore,mutation systems, frequently fortified with metabolic activation systems, are being used to screen for compoundswith potential electrophilic reactivity. The most common ofthese mutation systems utilizes the Salmonella typhimuriumtester strains devised by Ames and his associates (1), but awide variety of other bacteria, fungi, plants, insects, andmammalian cells are also being used (46, 79,167,197). Themutagenic activities of chemicals in the mammalian tissue-mediated bacterial systems have shown relatively goodqualitative correspondence with their carcinogenic activities for experimental animals and, where known, for humans (126,127,135,167,197,198). These assays, however,give some false-positive and some false-negative results;i.e., they have failed to show mutagenic activity for about10% of established carcinogens, especially some of theweaker ones, and they have shown mutagenic activity forsome chemicals that have thus far not induced tumors inanimal tests.

Assays have also been developed in which the end pointis the malignant transformation of mammalian cells inculture, and approaches to the fortification of these assaysystems with metabolic activation systems are being studied. The transformation assays are currently being evalu

ated for their abilities to predict the carcinogenic potentialsof chemicals, and preliminary results are encouraging (39,54, 160, 177). However, up to the present, and for theforeseeable future, none of these mutagenicity or transformation prescreens appears tojpe reliable enough to replacewhole animal carcinogenicity assays for the evaluation ofcarcinogenic activity.

Other approaches to the reduction of contact with ultimate carcinogens involve alteration of carcinogen metabolism so that less is converted to ultimate forms or enhancement of the intracellular levels of nucleophilic acceptorsthat can react in noncritical ways with the electrophilicultimate carcinogens. Experimental data have been collected for both of these approaches, and, as examinedespecially by Wattenberg (218), marked protection has beenachieved in a number of model systems in experimentalanimals. At present, it is difficult to explore these approaches in the human in view of the uncertainties of theramifications of such manipulations. Possible untowardeffects include an increased metabolic activation of somecarcinogens or increases in other toxic reactions (229).Further, some chemicals, such as phÃ©nobarbital, that haveinhibited tumor induction when administered simultaneously with a carcinogen, have shown promoting activitywhen applied subsequent to an initiating dose (158, 159).

Reduction of human hazard also requires knowledge ofwhere, how, and to what extent carcinogens are present inparticular places. We are surprised all too often by our lackof foresight. Thus, in 1950, many years after the carcinogenicity of 2-naphthylamine for the human bladder and itshazard in industrial situations was generally recognized,Case and Hosker (28) traced a cluster of cancers of theurinary bladder among rubber workers to 2-naphthylaminewhich was present as an impurity in the antioxidant thenused for curing rubber.

Study of the large group of carcinogenic W-nitroso compounds has provided similar evidence of the need for acuityin looking for potential exposures (147). An early concernwas the likelihood of formation of nitrosamines or nitrosam-ides in the stomach after ingestion of amines or amides inthe diet or as drugs together with nitrite (13,119). However,Tannenbaum, Preussmann, and their associates (193, 205)have now shown that the main source of stomach nitrite isdietary nitrate which is a normal constituent of many foods,especially certain vegetables. After absorption the nitrate isexcreted in the saliva and reduced by the bacteria in themouth; the nitrite thus formed reaches the stomach throughthe normal swallowing of saliva.

A/-Nitroso compounds have also been found as pollutants, fortunately usually at relatively low levels, in urban air,water, soil, and a variety of commercial products (13). Forinstance, Fan and his associates (47) have recently reportedconcentrations of 0.02 to 3% of A/-nitrosodiethanolamine,which induces liver tumors in the rat, in synthetic cuttingoils that contain triethanolamine and were placed in metalcans treated with nitrite to prevent corrosion. These synthetic cutting oils were introduced some years ago as areplacement for the mineral oil-based products that hadposed a risk of skin cancer to workmen. While the impactof these exposures on human cancer incidences is difficult to evaluate, it is apparent that we need much better





communication between those who formulate products andtheir packaging, those who have good knowledge of theadverse biological effects of chemicals, and those who canpredict the reactions that can be anticipated in givenmixtures of chemicals under a variety of conditions. Furthermore, astute clinical observations and careful epidemi-ological studies will continue to be important approachesfor assessing the success of our predictive and preventiveapproaches and for recognizing those situations that require further study.

Prevention of Promotion of Initiated Cells. Little workhas been directed toward the detection of promoting agentsthat may cause initiated cells to develop into gross tumors,although such chemicals must also occur in the humanenvironment. A very important approach to the modificationof promotion and reduction of cancer incidences appearsto be available through administration of certain retinoids.Vitamin A deficiency has long been known to result inhyperplasia and keratinization of epithelia, and Lasnitzski(111) some 20 years ago noted that high levels of vitamin Aprevented and even reversed the hyperplastic effects induced by 3-methylcholanthrene in organ cultures of mouse

prostates. Building on these and other results, Sporn,Bollag, and others (see Ref. 194) envisioned the synthesisof retinoids that can maintain differentiated epithelia in theface of carcinogenic insults, that lack the toxicity of highlevels of naturally occurring vitamin A, and that are retainedby extrahepatic tissues. The developments of the past fewyears are very promising. Reductions of the incidences ofcancers of the skin, lungs, urinary bladder, and breast inexperimental animals have been obtained even when theadministration of the retinoid was not begun until after thetreatment with the chemical carcinogen had been completed (194). Thus, through maintaining the differentiationof the epithelia previously exposed to the initiating activityof a chemical carcinogen, the retinoids are apparently ableto prevent some promoting influence associated with thehyperplastic state. The application of the synthetic retinoidÂ«to the inhibition of carcinogenesis in high-risk groups,

such as those known to have been exposed to urinarybladder carcinogens, is receiving serious consideration.

Concluding Remarks

It is apparent that there is need for much further researchto unlock the doors to a complete understanding of theprocesses involved in the induction of cancer. Nevertheless, a comparison of our state of knowledge today compared with that of 35, 25, or even 10 years ago encouragesme in the belief that further study will eventually bring adetailed insight into the reactions involved in the initiation,promotion, and progression of cells in the neoplastic process. This detailed knowledge will surely give us the bestfoundation for the prevention and possibly the treatment ofhuman cancers.

In the meantime, it is clear that the tremendous effort thathas gone into the study of experimental chemical carcinogenesis and into the epidemiology of human cancer overthe past 20 to 30 years has given us much informationrelevant to the prevention of cancer. Thus, these studieshave pinpointed certain industrial carcinogens, have clearly

indicated that each individual can markedly reduce hislikelihood of developing cancer by reducing his exposureto sunlight and cigarette smoke, and have given us methodsfor detecting potential human carcinogens. Much data haveclearly established that chemical carcinogenesis is astrongly dose-dependent phenomenon, although theshapes of the dose-response curves are rarely determined

over wide ranges and probably cannot be established forthe low exposures of interest to human populations. Thestrong dose dependence is too often slighted in publicdiscussions of possible human hazards from mutagenicand/or carcinogenic chemicals. While specific chemicalsare known to pose risks to certain human populations atrelatively high levels of exposure and others are suspectedof posing such risks, wide-spread prohibition of the use of

such chemicals at much lower levels may or may not bedesirable. Likewise, the extrapolations of carcinogenicitydata for experimental animals and of mutagenicity data toevaluations of possible hazards for human populations arevery difficult problems. The single fact that, at very highdoses, a chemical causes some cancers in experimentalanimals or some mutations may not be an adequate reasonfor removing it from uses beneficial to the public." The

questions to be asked must include how much risk a givenlow-level use may entail and how much benefit would be

lost to society from restriction on the use of that chemicalfor that purpose.

It is important that we recognize that life is a series ofrisks and benefits. I have been much impressed with thewritings by W. W. Lowrance on safety matters. Lowrance(117) defines safety "as a judgement of the acceptability ofrisk." Risk, in turn, he defines "as a measure of theprobability and severity of harm to human health." Heconcludes that a "thing is safe if its attendant risks arejudged to be acceptable." How to decide what acceptable

levels of risk are for each individual and for the populationas a whole are important societal problems that mustreceive much more attention. In highly industrialized societies the activities of each of us have some impact on othermembers of the society. At the same time rigid restrictionsof scientific, personal, societal, -and industrial activity,where they are not really needed for the common good, caneasily lead to important losses to society.

References

1. Ames, B. N., McCann, J., and Yamasaki, E. Methods for DetectingCarcinogens and Mutagens with the Salmonella/Mammalian-Micro-some Mutagenicity Test. Mutation Res., 31: 347-364, 1975.

4The recent report on the development of tumors of the urinary bladderin male mice fed the sweetener xylitol as 10 to 20% of their diets in 2-yeartests and its possible implications for the use of xylitol in human foodsprovide an example of the problems to be resolved (31). These tumorsapparently developed only in urinary bladders that contained stones (oxa-lates?), a condition long known to predispose rodents to the development ofbladder tumors. Neither bladder stones nor bladder tumors were reported infemale mice fed the high levels of xylitol or in male mice fed 2% of thesweetener. Since xylitol is a normal intermediate in the metabolism of D-glucuronate (Ref. 227, p. 491), has not shown mutagenic activity (8), andwould not be expected to yield strong electrophilic reactants on metabolism,there seems to be little basis for concern of hazard to humans ingesting lowlevels of xylitol in foods. Yet, strict interpretation of the Delaney amendmentto the Pure Food and Drug Act would prohibit the use of xylitol, since the actdoes not permit addition to food of any chemical that has caused tumors ineither humans or animals.

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E. C. Miller

2. Anderson, D. E. Familial Susceptibility. In: J. F. Fraumeni, Jr. (ed.),Persons at High Risk of Cancer: An Approach to Cancer Etiology andControl, pp. 39-54. New York: Academic Press, Inc., 1975. 31.

3. Armuth, V., and Berenblum, I. Systemic Promoting Action of Phorbol inLiver and Lung Carcinogenesis in AKR Mice. Cancer Res., 32: 2259- 32.

2262, 1972.4. Armuth, V., and Berenblum, I. Promotion of Mammary Carcinogenesis 33.

and Leukemogenic Action by Phorbol in Virgin Female Wistar Rats.Cancer Res., 34: 2704-2707, 1974. 34.

5. Barry, E. J., Malejka-Giganti, D., and Gutmann, H. R. Interaction ofAromatic Amines with Rat-Liver Proteins /'n Vivo. III. On the Mechanism

of Binding of the Carcinogens, W-2-Fluorenylacetamide and N-Hy- 35.droxy-2-fluorenylacetamide, to the Soluble Proteins. Chem.-Biol. Interactions, 1: 139-155, 1969.

6. Bartsch, H., Dworkin, M., Miller, J. A., and Miller, E. C. Electrophilic /Y- 36.Acetoxy-aminoarenes Derived from Carcinogenic A/-Hydroxy-A/-ace-tylaminoarenes by Enzymatic Deacetylation and Transacetylation inLiver. Biochim. Biophys. Acta, 286: 272-298, 1972. 37.

7. Bartsch, H., and Hecker, E. On the Metabolic Activation of the Carcinogen /V-Hydroxy-N-2-acetylaminofluorene. III. Oxidation with Horseradish Peroxidase to Yield 2-Nitrosofluorene and N-Acetoxy-N-2-acetyl-aminofluorene. Biochim. Biophys. Acta, 237: 567-578, 1971. 38.

8. Batzinger, R. P., Ou, S.-Y. L., and Bueding, E. Saccharin and OtherSweetners: Mutagenic Properties. Science, 798: 944-946, 1977.

9. Berenblum, I. The Cocarcinogenic Action of Croton Resin. Cancer 39.Res., 7: 44-48, 1941.

10. Berenblum, I. Carcinogenesis as a Biological Problem. 376 pp. Amsterdam: North-Holland Publishing Co., 1974. 40.

11. Berenblum, I., and Shubik, P. The Persistence of Latent Tumour CellsInduced in the Mouse's Skin by a Single Application of 9,10-Dimethyl-

1,2-benzanthracene. Brit. J. Cancer, 3: 384-386, 1949.12. Berwald, Y., and Sachs, L. In vitro Cell Transformation with Chemical 41.

Carcinogens. Nature, 200. 1182-1184, 1963.13. Bogovski, P., Walker, E. A., and Davis, W. /V-Nitroso Compounds in the

Environment. IARC Scientific Publication No. 9, 243 pp. Lyon, France: 42.International Agency for Research on Cancer, 1974.

14. Borgen, A., Darvey, H., Castagnoli, N., Crocker, T. T., Rasmussen, R. 43.E., and Wang, I. Y. Metabolic Conversion of Benzo(a)pyrene by SyrianHamster Liver Microsomes and Binding of Metabolites to Deoxyribo- 44.nucleic Acid. J. Med. Chem., 16: 502-506, 1973.

15. Bouck, N., and deMayorca, G. Somatic Mutation as the Basis for 45.Malignant Transformation of BHK Cells by Chemical Carcinogens.Nature, 264: 722-727, 1976.

16. Boutwell, R. K. Some Biological Aspects of Skin Carcinogenesis.Progr. Exptl. Tumor Res.. 4: 207-250, 1964. 46.

17. Boutwell, R. K. The Function and Mechanism of Promoters of Carcinogenesis. CRC Crit. Rev. Toxicol., 2: 419-443, 1974.

18. Boutwell, R. K. The Role of the Induction of Ornithine Decarboxylase in 47.Tumor Promotion. In: H. H. Hiatt, J. D. Watson, and J. A. Winsten(eds.), Origins of Human Cancer, Book B, pp. 773-783. Cold SpringHarbor, N. Y.: Cold Spring Harbor Laboratory, 1978. 48.

19. Boyland, E. The Biological Significance of Metabolism of PolycyclicCompounds. Biochem. Soc. Symp., 5: 40-54, 1950. 49.

20. Boyland, E., and Horning, E. S. The Induction of Tumours withNitrogen Mustards. Brit. J. Cancer, 3: 118-123, 1949. 50.

21. Boyland, E., and Sims, P. The Carcinogenic Activities in Mice ofCompounds Related to Benz(a)anthracene. Intern. J. Cancer, 2: 500- 51.

504, 1967.22. Braun, A. C. The Biology of Cancer, 169 pp. Reading, Mass.: Addison-

Wesley Publishing Co., 1974. 5223. Braun, A. C., and Wood, H. N. Suppression of the Neoplastic State with

the Acquisition of Specialized Functions Â¡nCells, Tissues, and Organsof Crown Gall Teratomas of Tobacco. Proc. Nati. Acad. Sei. U. S., 73: 53496-500, 1976.

24. Brookes, P., and Lawley, P. D. Evidence for the Binding of Polynuclear 54.Aromatic Hydrocarbons to the Nucleic Acids of Mouse Skin: Relationbetween Carcinogenic Power of Hydrocarbons and Their Binding toDeoxyribonucleic Acid. Nature, 202: 781-784, 1964.

25. Bryan, G. T., and Springberg, P. D. Role of the Vehicle in the Genesis 55of Bladder Carcinomas in Mice by the Pellet Implantation Technic.Cancer Res., 26: 105-109, 1966.

26. Burdette, W. J. The Significance of Mutation in Relation to the Originof Tumors: A Review. Cancer Res., 75: 201-226, 1955. 56

27. Butlin, H. T. Cancer of the Scrotum in Chimney-Sweeps and Others. II.Why Foreign Sweeps Do Not Suffer from Scrotal Cancer. Brit. Med. J.,2: 1-6, 1892.

28. Case, R. A. M., and Hosker, M. E. Tumour of the Urinary Bladder as an 57Occupational Disease in the Rubber Industry in England and Wales.Brit. J. Prevent. Soc. Med., 8: 39-50, 1954. 58

29. Clayson, D. B. Chemical Carcinogenesis, 467 pp. Boston: Little, Brownand Co., 1962.

30. Clayson, D. B., and Garner, R. C. Carcinogenic Aromatic Amines and 59Related Compounds. In: C. E. Searle (ed.), Chemical Carcinogens.

American Chemical Society Monograph No. 173, pp. 366-461. Washington, D. C.: American Chemical Society, 1976.Chemical and Engineering News. Xylitol Causes Some Cancer Â¡nMice.55: (No. 47): 7, 1977.Cleaver, J. E., and Bootsma, D. Xeroderma Pigmentosum: Biochemicaland Genetic Characteristics. Ann. Rev. Genet., 9: 19-38, 1975.Clemmesen, J. On the Etiology of Some Human Cancers. J. Nati.Cancer Inst., 72: 1-21, 1951.Cook, J. W., Duffy, E., and Schoental, R. Primary Liver Tumours inRats following Feeding with Alkaloids of Senecio Â¡acobaea. Brit. J.Cancer, 4: 405-410, 1950.Cook, J. W., Hewett, C. L., and Hieger, I. The Isolation of a Cancer-

Producing Hydrocarbon from Coal Tar. Parts I, II, and III. J. Chem.Soc., 395-405, 1933.Cramer, J. W., Miller, J. A., and Miller, E. C. N-Hydroxylation: A NewMetabolic Reaction Observed in the Rat with the Carcinogen 2-Acetyl-aminofluorene. J. Biol. Chem., 235: 885-888, 1960.DeBaun, J. R., Miller, E. C., and Miller, J. A. W-Hydroxy-2-acetylamino-

fluorene Sulfotransferase: Its Probable Role in Carcinogenesis andProtein-(methion-S-yl) Binding in Rat Liver. Cancer Res., 30: 577-595,

1970.DeBaun, J. R., Smith, J. Y. R., Miller, E. C., and Miller, J. A. Reactivityin Vivo of the Carcinogen /V-Hydroxy-2-acetylaminofluorene: Increaseby Sulfate Ion. Science, 767: 184-186, 1970.DiPaolo, J. A., Nelson, R. L.,and Donovan, P. J./n Vitro Transformationof Syrian Hamster Embryo Cells by Diverse Chemical Carcinogens.Nature, 235: 278-280, 1972.Dipple, A. Polynuclear Aromatic Carcinogens. In: C. E. Searle (ed.),Chemical Carcinogens. Amercian Chemical Society Monograph No.173, pp. 245-314. Washington, D. C.: American Chemical Society,1976.Doll, R. Prevention of Cancer â€”Pointers for Epidemiology. The RockCarling Fellowship 1967-Nuffield Provincial Hospitals Trust 1967.London: Whitefriars Press, Ltd., 1967.Doll, R. Strategy for Detection of Cancer Hazards to Man. Nature, 265:589-596, 1977.

Edwards, J. E. Hepatomas in Mice Induced with Carbon Tetrachloride.J. Nati. Cancer Inst., 2: 197-199, 1941.Emmett, E. A. Ultraviolet Radiation as a Cause of Skin Tumors. CRCCrit. Rev. Toxicol., 2: 211-255, 1973.Essigmann, J. M., Croy, R. G., Nadzan, A. M., Busby, W. F., Jr.,Reinhold, V. N., Buchi, G., and Wogan, G. N. Structural Identificationof the Major DNA Adduci Formed by Aflatoxin B, in Vitro. Proc. Nati.Acad. Sei. U. S., 74: 1870-1874, 1977.Fahmy, O. G., and Fahmy, M. J. Gene Elimination Â¡nCarcinogenesis:Reinterpretation of the Somatic Mutation Theory. Cancer Res., 30:195-205, 1970.

Fan, T. Y., Morrison, J., Rounbehler, D. P., Ross, R., Fine, D. H., Miles,W., and Sen, N. P. W-Nitrosodiethanolamine in Synthetic CuttingFluids: A Part-Per-Hundred Impurity. Science, 796: 70-71, 1977.Farber, E. Ethionine Carcinogenesis. Advan. Cancer Res., 7: 383-474,

1963.Farber, E. Carcinogenesis â€”Cellular Evolution as a Unifying Thread:Presidential Address. Cancer Res., 33: 2537-2550, 1973.Farber, E. Hyperplastic Liver Nodules. Methods Cancer Res., 7: 345-375, 1973.Farber, E., and Magee, P. N. The Probable Alkylation of Liver Ribonu-cleic Acid by the Hepatic Carcinogens Dimethylnitrosamine and Ethionine. Biochem. J., 76. 58P, 1960.Fischer, B. Die experimentelle Erzeungung atypischer Epithelwucherungen under die Entstehung bÃ¶sartiger GeschwÃ¼lste. Muench. Med.Wochschr., 53: 2041-2047, 1906.Fitzhugh, O. G., and Nelson, A. A. Liver Tumors in Rats Fed Thioureaor Thioacetamide. Science, 708: 626-628, 1948.Freeman, A. E., Weisburger, E. K., Weisburger, J. H., Wolford, R. G.,Maryak, J. M., and Huebner, R. J. Transformation of Cell Cultures asan Indication of the Carcinogenic Potential of Chemicals. J. Nati.Cancer Inst., 57: 799-807, 1973.Friedewald, W. F., and Rous, P. The Initiating and Promoting Elementsin Tumor Production. An Analysis of the Effects of Tar, Benzpyrene,and Methylcholanthrene on Rabbit Skin. J. Exptl. Med., 80: 101-126,1944.Fuchs, R. P. P., Lefevre, J. F., Pouyet, J., and Daune, M. P. Comparative Orientation of the Fluorene Residue in Native DNA Modified by N-Acetoxy-N-2-acetylaminofluorene and Two 7-Halogeno Derivatives.Biochemistry, 75: 3347-3351, 1976.Gardner, L. U., and Heslington, H. F. Osteosarcoma from IntravenousBeryllium Compounds in Rabbits. Federation Proc., 5: 221, 1946.Garner, R. C., Miller, E. C., and Miller, J. A. Liver Microsomal Metabolism of Aflatoxin B, to a Reactive Derivative Toxic to Salmonellatyphimurium TA 1530. Cancer Res., 32: 2058-2066, 1972.Goth, R., and Rajewsky, M. F. Persistence of O-6-Ethylguanine in Rat-Brain DNA: Correlation with Nervous System-Specific Carcinogenesis





by Ethylnitrosourea. Proc. Nati. Acad. Sei. U. S., 71: 639-643, 1974.60. Gross, L. Facts and Theories on Viruses Causing Cancer and Leukemia

Proc. Nati. Acad. Sei. U. S., 71: 2013-2017, 1974.61. Grover. P. L., Hewer, A., and Sims, P. Epoxides as Microsomal

Metabolites of Polycyclic Hydrocarbons. Federation European Bio-chem. Soc. Letters, 18: 76-80, 1971.

62. Grover, P. L., Sims, P., Huberman, E., Marquardt, H., Kuroki, T., andHeidelberger, C. In Vitro Transformation of Rodent Cells by K-RegionDerivatives of Polycyclic Hydrocarbons. Proc. Nati. Acad. Sei. U. S.,68. 1098-1101, 1971.

63. Gurdon. J. B. The Control of Gene Expression in Animal Development.160 pp. Cambridge, Mass.: Harvard University Press, 1974.

64. Haddow, A., and Kon, G. A. R. Chemistry of Carcinogenic Compounds.Brit. Med. Bull.,4: 314-326, 1947.

65. Haenszel, W. Migrant Studies. In: J. F. Fraumeni, Jr. (ed.), Persons atHigh Risk of Cancer. An Approach to Cancer Etiology and Control, pp.361-371. New York: Academic Press, Inc., 1975.

66. Haenszel, W., and Kurihara, M. Studies of Japanese Migrants. I.Mortality from Cancer and Other Diseases among Japanese in theUnited States. J. Nati. Cancer Inst., 40: 43-68, 1968.

67. Hall, W. H., and Bielschowsky, F. The Development of Malignancy inExperimental Induced Adenomata of the Thyroid. Brit. J. Cancer, 3:534-541,1949.

68. Hart, R. W., and Setlow, R. B. Direct Evidence That Pyrimidine Dimersin DNA Result in Neoplastic Transformation. In: P. C. Hanawalt and R.B. Setlow (eds.), Molecular Mechanisms for Repair of DNA, Part B, pp.719-724. New York: Plenum Press, 1975.

69. Hartwell, J. L. Survey of Compounds Which Have Been Tested forCarcinogenic Activity, Ed. 2, U. S. Public Health Service PublicationNo. 149, 583 pp. Washington, D. C.: U. S. Government Printing Office,1951.

70. Heath, C. W., Caldwell, G. G., and Feorino, P. C. Viruses and OtherMicrobes. In: J. F. Fraumeni, Jr. (ed.), Persons at High Risk of Cancer:An Approach to Cancer Etiology and Control, pp. 241-264. New York:Academic Press, Inc., 1975.

71. Hecker, E. Isolation and Characterization of the Cocarcinogenic Principles from Croton Oil. Methods Cancer Res., 6: 439-484, 1971.

72. Heidelberger, C. Studies on the Molecular Mechanism of HydrocarbonCarcinogenesis. J. Cellular Comp. Physiol . 64: (Suppl. 1): 129-148,1964.

73. Heidelberger, C. Chemical Oncogenesis in Culture. Advan. CancerRes., 78: 317-366, 1973.

74. Heidelberger, C. Chemical Carcinogenesis. Ann. Rev. Biochem., 44:79-121,1975.

75. Henry, S. A. Occupational Cutaneous Cancer Attributable to CertainChemicals in Industry. Brit. Med. Bull., 4: 389-401, 1947.

76. Hicks, R. M., Wakefield, J. St. J., and Chowaniec, J. Evaluation of aNew Model to Detect Bladder Carcinogens or Co-carcinogens; ResultsObtained with Saccharin, Cyclamate, and Cyclophosphamide. Chem.Biol. Interactions, 11: 225-233, 1975.

77. Hieger, I. LVIII. The Spectra of Cancer-Producing Tars and Oils ofRelated Substances. Biochem. J., 24: 505-511, 1930.

78. Higginson, J. Present Trends in Cancer Epidemiology. Can. CancerConf.,8: 40-75, 1969.

79. Hollaender, A. (ed.). Chemical Mutagens: Principles and Methods forTheir Detection. Vols. 1 to 3. New York: Plenum Press, 1971-1973.

80. Huberman, E., Kuroki, T., Marquardt, H., Selkirk, J. K., Heidelberger,C., Grover, P. L., and Sims, P. Transformation of Hamster EmbryoCells by Epoxides and Other Derivatives of Polycyclic Hydrocarbons.Cancer Res., 32: 1391-1396, 1972.

81. Huberman, E., Sachs, L., Yang, S. K., and Gelboin, H. V. Identificationof Mutagenic Metabolites of Benzo(a)pyrene in Mammalian Cells. Proc.Nati. Acad. Sei. U. S., 73: 607-611, 1976.

82. Hueper, W. C., Wiley, F. H., and Wolfe, H. D. ExpÃ©rimentalProductionof Bladder Tumors in Dogs by Administration of Beta-naphthylamine.J. Ind. Hyg. Toxicol., 20: 46-84. 1938.

83. Hughes, P. E. The Significance of Staining Reactions of PreneoplasticRat Liver with Fluorescein-Globulin Complexes. Cancer Res., 18: 426-432, 1958.

84. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicalsto Man. Vols. 1 to 15. Lyon, France: International Agency for Researchon Cancer, 1972-1977.

85. Illmensee, K., and Mintz, B. Totipotency and Normal Differentiation ofSingle Teratocarcinoma Cells Cloned by Injection into Blastocysts.Proc. Nati. Acad. Sei. U. S., 73: 549-553, 1976.

86. Irving, C. C. Conjugates of W-Hydroxy Compounds. In: W. H. Fishman(ed.), Metabolic Conjugation and Metabolic Hydrolysis, pp. 53-119.New York: Academic Press, Inc.. 1970.

87. Irving, C. C., Janss, D. H., and Russell, L. T. Lack of N-Hydroxy-2-acetylaminofluorene Sulfotransferase Activity in the Mammary Glandand Zymbal's Gland of the Rat. Cancer Res., 31: 387-391, 1971.

88. Jablon. S. Radiation. In: J. F. Fraumeni, Jr. (ed.), Persons at High Riskof Cancer: An Approach to Cancer Etiology and Control, pp. 151-165.

New York: Academic Press, Inc., 1975.89. Jacob, F., and Monod, J. Genetic Regulatory Mechanisms in the

Synthesis of Proteins. J. Mol. Biol.,3: 318-356, 1961.90. Jerina. D. M., Yagi, H.. Hernandez. O.. Dansette, P. M.. Wood. A. W.,

Levin, W., Chang, R. L., Wislocki, P. G., and Conney. A. H. Synthesisand Biological Activity of Potential Benzo(a)pyrene Metabolites. In R.Freudenthal and P. W. Jones (eds.), Carcinogenesis-A Comprehensive Survey, Vol. 1. Polynuclear Aromatic Hydrocarbons: Chemistry,Metabolism, and Carcinogenesis, pp. 91-113. New York: Raven Press,1976.

91. John I. Thompson and Co. Survey of Compounds Which Have BeenTested for Carcinogenic Activity, Vol. 1968-1969. U. S. Public HealthService Publication No. 149. Washington, D. C.: U. S. GovernmentPrinting Office, 1972.

92. Kadlubar, F. F., Miller, J. A., and Miller, E. C. Microsomal N-Oxidationof the Hepatocarcinogen /V-Methyl-4-aminoazobenzene and the Reactivity of W-Hydroxy-W-methyl-4-aminoazobenzene. Cancer Res., 36:1196-1206, 1976.

93. Kadlubar, F. F., Miller, J. A., and Miller, E. C. Hepatic Metabolism of N-Hydroxy-N-methyl-4-aminoazobenzene and Other N-Hydroxyarylam-ines to Reactive Sulfuric Acid Esters. Cancer Res., 36: 2350-2359,1976.

94. Kadlubar, F. F., Miller, J. A., and Miller, E. C. Hepatic Microsomal N-Glucuronidation and Nucleic Acid Binding of /V-Hydroxy Arylamines inRelation to Urinary Bladder Carcinogenesis. Cancer Res., 37: 805-814,1977.

95. Kapitulnik, J., Levin, W., Conney, A. H., Yagi, H., and Jerina, D. M.Benzo(a)pyrene 7,8-Dihydrodiol Is More Carcinogenic ThanBenzo(a)pyrene in Newborn Mice. Nature, 266: 378-380, 1970.

96. Kennaway, E. L. Experiments on Cancer-Producing Substances. Brit.Med. J.,2: 1-4, 1925.

97. Kennaway, E. L., and Hieger, I. Carcinogenic Substances and TheirFluorescence Spectra. Brit. Med. J., 1: 1044-1046, 1930.

98. Ketterer, B., Tipping, E., Beale, D. Meuwissen, J., and Kay, C. M.Proteins Which Specifically Bind Carcinogens. In: P. Bucalossi. U.Veronesi, and N. Cascinelli (eds.). Proceedings of the Eleventh International Cancer Congress, Vol. 2, Chemical and Viral Carcinogenesis,pp. 25-29. Amsterdam: Excerpta Medica, 1975.

99. King, C. M. Mechanism of Reaction, Tissue Distribution, and Inhibitionof Arylhydroxyamic Acid Acyltransferase. Cancer Res., 34: 1503-1515,1974.

100. King, C. M., and Olive, C. W. Comparative Effects of Strain, Speciesand Sex on the Acyltransferase- and Sulfotransferase-catalyzed Activations of N-Hydroxy-/V-2-fluorenylacetamide. Cancer Res., 35: 906-912,1975.

101. King, C. M., and Phillips, B. Enzyme-catalyzed Reactions of theCarcinogen N-Hydroxy-2-fluorenylacetamide with Nucleic Acid. Science, 159: 1351-1353, 1968.

102. Kinosita, R. Researches on the Carcinogenesis of the Various ChemicalSubstances. Gann, 30: 423-426, 1936.

103. Kipling, M. D. Soots, Tars, and Oils as Causes of Occupational Cancer.In: C. E. Searle (ed.), Chemical Carcinogens. American ChemicalSociety Monograph No. 173, pp. 315-323. Washington, D. C.: AmericanChemical Society, 1976.

104. Knudson, A. G., Jr. Genetic Influences in Human Tumors. In: F. F.Becker (ed.). Cancer: A Comprehensive Treatise, Vol. 1, pp. 59-74.New York: Plenum Press, 1975.

105. Koreeda, M., Moore, P. D., Yagi, H., Yeh, H. J. C., and Jerina, D. M.Alkylation of Polyguanylic A~'H at the 2-Amino Group and Phosphateby the Potent Mutagen (Â±)-7/3,8a-Dihydroxy-9/3,10/3-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. J. Am. Chem. Spc.. 98. 6720-6722. 1976.

106. Kriek, E. Persistent Binding of a New Reaction Product of the Carcinogen N-Hydroxy-2-acetylaminofluorene with Guanine in Rat Liver DNAin Vivo. Cancer Res., 32: 2042-2048, 1972.

107. Kriek, E. Carcinogenesis by Aromatic Amines. Biochem. Biophys. Acta,355: 177-203,1974.

108. Kuroki, T., Huberman, E., Marquardt, H. Selkirk. J. K., Heidelberger,C., Grover, P. L., and Sims, P. Binding of K-Region Epoxides andOther Derivatives of Benz(a)anthracene and Dibenz(a,h)anthracene toDNA, RNA, and Proteins of Transformable Cells. Chem.-Biol. Interactions, 4: 398-397, 1971.

109. Lacassagne, A. Apparition de Cancers de la Mamelle chez la SourisMÃ¢le,soumise Ã des Injections de Folliculine. Compi. Rend., 795: 630-632, 1932.

110. Lasne, C., Gentil, A., and Chouroulinkov, I. Two-Stage MalignantTransformation of Rat Fibroblasts in Tissue Culture. Nature. 247: 490-491, 1974.

111. Lasnitzki, I. The Influence of A Hypervitaminosis on the Effect of 20-Methylcholanthrene on Mouse Prostate Glands Grown in Vitro. Brit. J.Cancer, 9: 434-441, 1955.

112. Lawley, P. D. Carcinogenesis by Alkylating Agents. In: C. E. Searle(ed.), Chemical Carcinogens. American Chemical Society MonographNo. 173, pp. 83-244. Washington, D. C.: American Chemical Society,

JUNE 1978 1493



E. C. Miller

1976113. Levin. W.. Wood, A. W.. Chang. R. L., Slaga, T. J., Yagi. H., Jerina. D.

M., and Conney, A. H. Marked Differences in the Tumor-initiatingActivity of Optically Pure (+)- and (-)-frans-7,8-Dihydroxy-7,8-dihydro-benzo(a)pyrene on Mouse Skin. Cancer Res.. 37. 2721-2725, 1977.

114. Lin. J.-K.. Miller, J. A., and Miller, E. C. Structures of Hepatic NucleicAcid-Bound Dyes in Rats Given the Carcinogen W-Methyl-4-aminoazo-benzene. Cancer Res., 35 844-850, 1975.

115. Lin, J.-K., Miller. J. A., and Miller, E. C. 2,3-Dihydro-2-(guan-7-yl)-3-hydroxyaflatoxin B,, a Major Acid Hydrolysis Product of Aflatoxin B,-DNA or -rRNA Adducts Formed in Hepatic Microsome-mediated Reactions and in Rat Liver in Vivo. Cancer Res.. 37. 4430-4438, 1977.

116. Loveless, A. Possible Relevance of 0-6 Alkylation of Deoxyguanosineto the Mutagenicity and Carcinogenicity of Nitrosamines and Nitrosam-ides. Nature, 223 206-207, 1969.

117. Lowrance, W. W. Of Acceptable Risk: Science and the Determinationof Safety, 180 pp. Los Altos, Calif.: William Kaufmann, Inc., 1976.

118 Magee. P. N., and Farber. E. Toxic Liver Injury and CarcinogenesisMethylation of Rat-Liver Nucleic Acids by Dimethylnitrosamine/n Vivo.Biochem. J.,83: 114-124. 1962.

119. Magee, P. N.. Montesano, R.. and Preussmann. R. /V-Nitroso Compounds and Related Carcinogens. In: C. E. Searle (ed.), ChemicalCarcinogens. American Chemical Society Monograph No. 173, pp.491-625. Washington, D. C.: American Chemical Society, 1976.

120. MÃ¤her.V. M.. Miller. E. C.. Miller. J. A., and Szybalski. W. Mutationsand Decreases in Density of Transforming DNA Produced by Derivativesof the Carcinogens 2-Acetylaminofluorene and /V-Methyl-4-aminoazo-benzene. Mol. Pharmacol., 4: 411-426, 1968.

121. Mainigi, K. D., and Sorof, S. Evidence for a Receptor Protein ofActivated Carcinogen. Proc. Nati. Acad. Sei. U. S., 74: 2293-2296,1977.

122. Marquardt. H., Baker, S.. Grab. D., and Marquardt. H. Oncogenic andMutagenic Activity of Adriamycin Decreased by Microsomal Metabolism. Proc. Am. Assoc. Cancer Res., 18: 13, 1977.

123. Marroquin, R F , and Farber. E. The Apparent Binding of Radioactive2-Acetylaminofluorene to Rat-Liver Ribonucleic Acid in Vivo Biochim.Biophys. Acta, 55. 403-405. 1962.

124 Martin. C. N.. and Garner, R. C. Aflatoxin B,-oxide Generated byChemical or Enzymic Oxidation of Aflatoxin B, Causes Guanine Substitution in Nucleic Acids. Nature, 267: 863-865, 1977.

125. Matsushima, T., Kakizoe, T., Kawachi. T., HarÃ¡, K., Sugimura, T.,Takeuchi, T.. and Umezawa, H. Effects of Protease-lnhibitors of Micro-bial Origin on Experimental Carcinogenesis. In: P. N. Magee, S.Takayama, T. Sugimura, and T. Matsushima (eds.), Fundamentals inCancer Prevention, pp. 57-68. Baltimore: University Park Press, 1976.

126. McCann. J., and Ames, B. N. Detection of Carcinogens as Mutagens inthe Sa/monella Microsome Test: Assay of 300 Chemicals. Discussion.Proc. Nati. Acad. Sei. U. S.. 73: 950-954. 1976.

127. McCann. J., Choi, E.. Yamasaki, E., and Ames, B. N. The Detection ofCarcinogens as Mutagens in the Salmonella Microsome Test: Assay of300 Chemicals. Proc Nati. Acad. Sei. U. S., 72. 5135-5139, 1975.

128. McKinnell, R. G.. Deggins, B. A., and Labat, D. D. Transplantation ofPluripotential Nuclei from Triploid Frog Tumors. Science, 765. 394-396, 1969.

129. Meehan. T.. StrÃ¤ub,K., and Calvin. M. Benzo(a)pyrene Diol EpoxideCovalently Binds to Deoxyguanosine and Deoxyadenosine in DNA.Nature, 269. 725-727, 1977.

130. Miller. E. C., Lotlikar, P. D., Miller, J. A., Butler, B. W.. Irving C. C., andHill, J. T. Reactions in Vitro of Some Tissue Nucleophiles with theGlucuronide of the Carcinogen N-Hydroxy-2-acetylaminofluorene. Mol.Pharmacol., 4: 147-154, 1968.

131. Miller. E. C , and Miller, J. A. The Presence and Significance of BoundAminoazo Dyes in the Livers of Rats Fedp-Dimethylaminoazobenzene.Cancer Res., 7. 468-480, 1947.

132. Miller, E. C., and Miller, J. A. In Vivo Combinations between Carcinogens and Tissue Constituents and Their Possible Role in Carcinogenesis. Cancer Res.. 72. 547-556, 1952.

133. Miller, E. C..and Miller. J. A. Mechanisms of Chemical Carcinogenesis:Nature of Proximate Carcinogens and Interactions with Macromole-cules. Pharmacol. Rev.. 78. 805-838, 1966.

134. Miller, E. C., and Miller. J. A. Low Carcinogenicity of the K-RegionEpoxides of 7-Methylbenz(a)anthracene and Benz(a)anthracene in theMouse and Rat. Proc. Soc. Exptl. Biol. Med.. 124: 915-919, 1967.

135. Miller, E. C., and Miller, J. A. The Mutagenicity of Chemical Carcinogens: Correlations. Problems, and Interpretations. In: A. Hollaender(ed.). Chemical Mutagens â€”Principles and Methods for Their Detection, Vol. 1, pp. 83-119. New York: Plenum Press, 1971.

136. Miller, E. C.. Miller, J. A., and Enomoto, M. The Comparative Carcino-genicities of 2-Acetylaminofluorene and Its W-Hydroxy Metabolite inMice, Hamsters, and Guinea Pigs. Cancer Res., 24: 2018-2032, 1964.

137. Miller, E. C., Miller, J. A., and Hartmann, H. A. N-Hydroxy-2-acetylami-nofluorene: A Metabolite of 2-Acetylaminofluorene with Increased Carcinogenic Activity in the Rat. Cancer Res., 27; 815-824, 1961.

138. Miller, E. C., Miller, J. A., Sandin, R. B.. and Brown, R. K. TheCarcinogenic Activities of Certain Analogues of 2-Acetylaminofluorenein the Rat. Cancer Res., 9. 504-509, 1949.

139. Miller. E. C., Sandin, R. B., Miller, J. A., and Rusch, H. P. TheCarcinogenicity of Compounds Related to 2-Acetylaminofluorene. III.Aminobiphenyl and Benzidine Derivatives. Cancer Res., 76; 525-534,1956.

140. Miller. J. A., and Miller. E. C. The Metabolic Activation of CarcinogenicAromatic Amines and Amides. Progr. Exptl. Tumor Res.. 77: 273-301,1969.

141. Miller, J. A., and Miller. E. C. Metabolic Activation of CarcinogenicAromatic Amines and Amides via N-Hydroxylation and N-HydroxyEsterification and Its Relationship to Ultimate Carcinogens as Electro-philic Reactants. In: E. D. Bergmann and B. Pullman (eds.). TheJerusalem Symposia on Quantum Chemistry and Biochemistry, Physi-cochemical Mechanisms of Carcinogenesis, Vol. 1, pp. 237-261. Jerusalem: The Israel Academy of Sciences and Humanities, 1969.

142. Miller, J. A., and Miller, E. C. Carcinogens Occurring Naturally inFoods. Federation Proc.,35: 1316-1321. 1976.

143. Miller, J. A., Sandin, R. B., Miller. E. C.. and Rusch, H. P. TheCarcinogenicity of Compounds Related to 2-Acetylaminofluorene. II.Variations in the Bridges and the 2-Substituent. Cancer Res., 75: 188-199, 1955.

144. Millette. R. L., and Fink, L. M. The Effect of Modification of T7 DNA bythe Carcinogen A/-2-Acetylaminofluorene: Termination of Transcriptionin Vitro. Biochemistry, 14: 1426-1432, 1975.

145. Mintz, B., and Illmensee, K. Normal Genetically Mosaic Mice Producedfrom Malignant Teratocarcinoma Cells. Proc. Nati. Acad. Sci. U.S., 72:3583-3589, 1975.

146. Mirvish, S. S. The Carcinogenic Action and Metabolism of Urethan and/V-Hydroxyurethan. Advan. Cancer Res., 11: 1-42, 1968.

147. Mirvish, S. S. Formation of /V-Nitroso Compounds: Chemistry, Kinetics,and in Vivo Occurrence. Toxicol. Appi. Pharmacol., 37: 325-351, 1975.

148. Mondai. S.. Brankow. D. W.. and Heidelberger. C. Two-Stage ChemicalOncogenesis in Cultures of C3H/10T'/2 Cells. Cancer Res., 36: 2254-2260. 1976.

149. Moschel. R. C., Baird. W. M.. and Dipple, A Metabolic Activation of theCarcinogen 7,12-Dimethylbenz(a)anthracene for DNA Binding. Biochem Biophys. Res. Commun., 76: 1092-1098, 1977.

150. Mottram, J. C. A Developing Factor in Experimental Blastogenesis. J.Pathol. Bacteriol., 56: 181-187, 1944.

151. Muir, C. S., and Kirk, R. Betel, Tobacco, and Cancer of the Mouth.Brit. J. Cancer, 14: 597-608, 1960.

152. Mulvihill, J. J. Congenital and Genetic Diseases. In: J. F. Fraumeni, Jr.(ed.). Persons at High Risk of Cancer: An Approach to Cancer Etiologyand Control, pp. 3-35. New York: Academic Press, Inc., 1975.

153. Nettleship, A., and Henshaw, P. S. Induction of Pulmonary Tumors inMice with Ethyl Carbamate (Urethane). J. Nati. Cancer Inst., 4: 309-319, 1943.

154. Nowinski. R. C., and Miller, E. C. Endogenous Oncornaviruses inChemically Induced Transformation. II. Effect of Virus Production inVivo. J. Nati Cancer Inst., 57: 1347-1350. 1976.

155. O'Brien, T. G., Simsiman, R. C., and Boutwell, R. K. Induction of thePolyamine-Biosynthetic Enzymes in Mouse Epidermis and Their Specificity for Tumor Promotion. Cancer Res., 35: 2426-2433, 1975.

156. Osborne, M. R., Beland, F. A., Harvey, R. G., and Brookes, P. Reactionof (Â±)-7a,8/3-Dihydroxy-9/3,10/3-Epoxy-7,8,9,10-tetrahydrobenzo(a)pyrenewith DNA. Intern. J. Cancer, 78: 362-368, 1976.

157. Pegg, A. E. Formation and Metabolism of Alkylated Nucleosides:Possible role in Carcinogenesis by Nitroso Compounds and AlkylatingAgents. Advan. Cancer Res., 25: 195-269, 1977.

158. Peraino, C., Fry, R. J. M., and Staffeldt, E. Reduction and Enhancementby PhÃ©nobarbitalof Hepatocarcinogenesis Induced in the Rat by 2-Acetylaminofluorene. Cancer Res.,37: 1506-1512, 1971.

159. Peraino, C., Fry, R. J. M., Staffeldt. E., and Kisieleski, W. E. Effects ofVarying the Exposure to PhÃ©nobarbital on Its Enhancement of 2-Acetylaminofluorene-lnduced Hepatic Tumorigenesis in the Rat. Cancer Res., 33: 2701-2705, 1973.

160. Pienta, R. J., Poiley, J. A., and Lebherz, W. B., III. MorphologicalTransformation of Early Passage Golden Syrian Hamster Embryo CellsDerived from Cryopreserved Primary Cultures as a Reliable in VitroBioassay for Identifying Diverse Carcinogens. Intern. J. Cancer, 79-642-655, 1977.

161. Pierce, G. B. Differentiation of Normal and Malignant Cells. FederationProc., 29: 1248-1254, 1970.

162. Pilot, H. C. The Natural History of Neoplasia. Newer Insights into anOld Problem. Am. J. Pathol., 89: 401-411, 1977.

163. Pilot, H. C., and Heidelberger, C. Metabolic Regulatory Circuits andCarcinogenesis. Cancer Res.,23 1694-1700. 1963.

164. Pott, P. Chirurgical Observations Relative to the Cancer of the Scrotum.London, 1775. Reprinted in Nati. Cancer Inst. Monograph, 70' 7-131963.

165. Price, P. J., Bellew, T. M., King, M. P., Freeman, A. E., Gilden, R. V.,




and Huebner, R. J. Prevention of Viral-Chemical Co-carcinogenesis inVitro by Type-specific Anti-viral Antibody. Proc. Nati. Acad. Sei. U. S.,73: 152-155, 1976.

166. Pullman, A., and Pullman, B. Electronic Structure and CarcinogenicActivity of Aromatic Molecules. Advan. Cancer Res., 3. 117-169, 1955.

167. Purchase, I. F. H., Longstaff, E., Ashby, J.. Styles. J. A., Anderson, D.,Lefevre, P. A., and Westwood, F. R. Evaluation of Six Short Term Testsfor Detecting Organic Chemical Carcinogens and Recommendationsfor Their Use. Nature, 264: 624-627, 1976.

168. Purves, H. D., and Griesbach, W. E. Studies on Experimental Goitre.VIII. Thyroid Tumours in Rats Treated with Thiourea. Brit. J. Exptl.Pathol.,28: 46-53, 1947.

169. Radomski, J. L., and Brill, E. Bladder Cancer Induction by AromaticAmines: Role of N-Hydroxy Metabolites. Science, 167: 992-993, 1970.

170. Radomski, J. L., Hearn, W. L., Radomski, T., Moreno, H., and Scott,W. E. Isolation of the Glucuronic Acid Conjugate of N-Hydroxy-4-aminobiphenyl from Dog Urine and Its Mutagenic Activity. Cancer Res.,37: 1757-1762, 1977.

171. Rapp, U. R., Nowinski, R. C., Reznikoff, C. A., and Heidelberger, C.Endogenous Oncornaviruses in Chemically Induced Transformation. I.Transformation Independent of Virus Production. Virology, 65: 392-409, 1975.

172. Redmond, D. E., Jr. Tobacco and Cancer: The First Clinical Report,1761. New Engl. J. Med., 282: 18-23, 1970.

173. Rehn, L. BlasengeschwÃ¼lstebei Fuchsin-Arbeitern. Arch. Klin. Chir.,50: 588-600, 1895.

174. Reich, E., Rifkin, D. B., and Shaw, E. (eds.). Proteases and BiologicalControl, 1021 pp. Cold Spring Harbor, N. Y.: Cold Spring HarborLaboratory, 1975.

175. Reitz, M. S., Saxinger, W. C., Ting, R. C., Gallo, R. C., and DiPaolo, J.A. Lack of Expression of Type C Hamster Virus after NeoplasticTransformation of Hamster Embryo Fibroblasts by Benzo(a)pyrene.Cancer Res., 37: 3585-3589, 1977.

176. Reznikoff, W. S. The Operon Revisited. Ann. Rev. Genet., 6: 133-156,1972.

177. Rhim, J. S., Park, D. K., Weisburger, E. K., and Weisburger, J. H.Evaluation of an in Vitro Assay System for Carcinogens Based on PriorInfection of Rodent Cells with Nontransforming RNA Tumor Virus. J.Nati. Cancer Inst., 52: 1167-1173, 1974.

178. Rossi, L., Ravera, M., Repetti, G., and Santi, L. Long-term Administration of DDT or Phenobarbital-Na in Wistar Rats. Intern. J. Cancer, 79:179-185, 1977.

179. Rous, P., and Kidd, J. G. Conditional Neoplasms and Sub-thresholdNeoplastic States: A Study of the Tar Tumors of Rabbits. J. Exptl. Med.,73:365-390,1941.

180. Salaman, M. H., and Roe, F. J. C. Incomplete Carcinogens: EthylCarbamate (Urethane) as an Initiator of Skin Tumor Formation in theMouse. Brit. J. Cancer, 7: 472-481, 1953.

181. Scherer, E., and Emmelot, P. Foci of Altered Liver Cells Induced by aSingle Dose of Diethylnitrosamine and Partial Hepatectomy: TheirContribution to Hepatocarcinogenesis in the Rat. European J. Cancer,11: 145-154, 1975.

182. Scherer, E., and Emmelot, P. Kinetics of Induction and Growth ofPrecancerous Liver-Cell Foci, and Liver Tumour Formation by Diethylnitrosamine in the Rat. European J. Cancer, 11: 689-696. 1975.

183. Schoental, R. Carcinogens in Plants and Micro-organisms. In: C. E.Searle (ed.). Chemical Carcinogens. American Chemical Society Monograph No. 173, pp. 626-689. Washington, D. C.: American ChemicalSociety, 1976.

184. Selkirk, J. K., Huberman, E., and Heidelberger, C. An Epoxide Is anIntermediate in the Microsomal Metabolism of the Chemical Carcinogen, Dibenz(a,h)anthracene. Biochem. Biophys. Res. Commun., 43:1010-1016,1971.

185. Shubik, P., and Hartwell, J. L. Survey of Compounds Which Have BeenTested for Carcinogenic Activity, Suppl. 1 and 2. U. S. Public HealthService Publication No. 149. Washington, D. C.: U. S. GovernmentPrinting Office, 1957, 1969.

186. Sims, P. The Carcinogenic Activities in Mice of Compounds Related to3-Methycholanthrene. Intern. J. Cancer, 2: 505-508, 1967.

187. Sims, P., and Grover. P. L. Epoxides in Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis. Advan. Cancer Res.,20. 166-274,1974.

188. Sims, P., Grover, P. L., Swaisland, A., Pal, K., and Hewer, A. MetabolicActivation of Benzo(a)pyrene Proceeds by a Diol-Epoxide. Nature, 252.326-328, 1974.

189. Singer, B. The Chemical Effects of Nucleic Acid Alkylation and TheirRelation to Mutagenesis and Carcinogenesis. Progr. Nucleic Acid Res.Mol. Biol., 15: 219-284, 1975.

190. Singer, B. All Oxygens in Nucleic Acids React with CarcinogenicEthylating Agents. Nature, 264: 333-339, 1976.

191. Smoking and Health. Report of the Advisory Committee to the SurgeonGeneral of the Public Health Service. Public Health Service PublicationNo. 1103, pp. 149-196. Washington, D. C.: U. S. Government Printing


Office, 1964.192. Sorof, S., and Young, E. M. Soluble Cytoplasmic Macromolecules of

Liver and Liver Tumor. Methods Cancer Res., 3: 467-548, 1967193. Spiegelhalder, B.. Eisenbrand, G., and Preussmann, R. Influence of

Dietary Nitrate on Nitrite Content of Human Saliva: Possible Relevanceto/n Vivo Formation of /V-Nitroso Compounds. Food Cosmet. Toxicol..14: 545-548, 1976.

194. Sporn, M. B., Dunlop. N. M.. Newton. D. L , and Smith, J. M. PrÃ©ventionof Chemical Carcinogenesis by Vitamin A and Its Synthetic Analogs(Retinoids). Federation Proc.,35. 1332-1338, 1976.

195. Stekol, J. A., Mody, U.. and Perry, J. The Incorporation of the Carbonof the Ethyl Group of Ethionine into Liver Nucleic Acids and the Effectof Ethionine Feeding on the Content of Nucleic Acids in Rat Liver. J.Biol. Chem.,235: PC59-PC60, 1960.

196. Stier, A., Reitz, I., and Sackmann, E. Radical Accumulation in LiverMicrosomal Membranes during Biotransformation of Aromatic Aminesand Nitro Compounds. Exptl. Arch. Pathol. Pharmakol., 274: 189-194,1972.

197. Stoltz, D. R., Poirier, L. A., Irving, C. C., Stich, H. F., Weisburger, J. H.,and Grice, H. C. Evaluation of Short-term Tests for Carcinogenicity.Toxicol. Appi. Pharmacol., 29: 157-180, 1974.

198. Sugimura, T., Sato, S., Nagao, M., Yahagi, T., Matsushima, T., Seino,Y., Takeuchi, M., and Kawachi, T. Overlapping of Carcinogens andMutagens. In: P. N. Magee, S. Takayama, T. Sugimura, and T. Matsushima (eds.), Fundamentals in Cancer Prevention, pp. 191-213. Baltimore: University Park Press, 1976.

199. Swaisland, A. J., Hewer, A., Pal, K., Keysell, G. R., Booth, J., Grover,P. L., and Sims, P. Polycyclic Hydrocarbon Epoxides: The Involvementof 8,9-Dihydro-8,9-dihydroxybenz(a)anthracene 10,11-Oxide in Reactions with the DNA of Benz(a)anthracene-treated Hamster EmbryoCells. Federation European Biochem. Soc. Letters, 47: 34-38, 1974.

200. Swenson, D. H., Lin, J.-K., Miller, E. C., and Miller, J. A. Aflatoxin B,-2,3-oxide as a Probable Intermediate in the Covalent Binding ofAflatoxins B, and B: to Rat Liver DNA and Ribosomal RNA in VivoCancer Res.,37: 172-181, 1977.

201. Swenson. D. H., Miller, E. C., and Miller, J. A. Aflatoxin B,-2,3-oxide:Evidence for Its Formation in Rat Liver in Vivo and by Human LiverMicrosomes in Vitro. Biochem. Biophys. Res. Commun., 60: 1036-1043,1974.

202. Swenson, D. H., Miller, J. A., and Miller, E. C. 2,3-Dihydro-2,3-dihy-droxy-aflatoxin B,: An Acid Hydrolysis Product of an RNA-Aflatoxin B,Adduci Formed by Hamster and Rat Liver Microsomes in Vitro. Biochem. Biophys. Res. Commun., 53: 1260-1267, 1973.

203. Swenson, D. H., Miller, J. A., and Miller, E. C. The Reactivity andCarcinogenicity of Aflatoxin BI-2,3-dichloride, a Model for the Putative2,3-Oxide Metabolite of Aflatoxin B,. Cancer Res.,35. 3811-3823, 1975.

204. Tada, M., and Tada, M. Metabolic Activation of 4-Nitroquinoline 1-Oxide and Its Binding to Nucleic Acid. In: P. N. Magee, S. Takayama,T. Sugimura, and T. Matsushima (eds.), Fundamentals of CancerPrevention, pp. 217-227. Baltimore: University Park Press. 1976.

205. Tannenbaum, S. R., Weisman, M., and Fett, D. The Effect of NitrateIntake on Nitrite Formation in Human Saliva. Food Cosmet. i'oxicol .

14: 549-552. 1976.206. Tasseron, J. G., Diringer. H., Frowirth, N., Mirvish, S. S., and Heidel

berger, C. Partial Purification of Soluble Protein from Mouse Skin toWhich Carcinogenic Hydrocarbons Are Specifically Bound. Biochemistry, 9: 1636-1644, 1970.

207. Tierney, B., Hewer, A., Walsh, C., Grover, P. L., and Sims, P. TheMetabolic Activation of 7-Methylbenz(a)anthracene in Mouse Skin.Chem.-Biol. Interactions, 18: 179-193, 1977.

208. Tracor/Jitco. Survey of Compounds Which Have Been Tested forCarcinogenic Activity, Vols. 1961-1967, 1970-1971, 1972-1973. U. S.Public Health Service Publication No. 149. Washington, D. C.: U. S.Government Printing Office. 1973, 1973, 1975.

209. Troll, W. Blocking Tumor Promotion by Protease Inhibitors. In: P. N.Magee, S. Takayama, T. Sugimura, and T. Matsushima (eds.), Fundamentals in Cancer Prevention, pp. 41-55. Baltimore: University ParkPress, 1976.

210. Trosko, J. E., and Chu, E. H. Y. The Role of DNA Repair and SomaticMutation in Carcinogenesis. Advan. Cancer Res.. 21: 391-425, 1975.

211. Turgeon, R., Wood, H. N., and Braun, A. C. Studies on the Recovery ofCrown Gall Tumors. Proc. Nati. Acad. Sei. U. S., 73: 3562-3564, 1976.

212. Urbach, F., Rose, D. B., and Bonnern, M. Genetic and EnvironmentalInteractions in Skin Carcinogenesis. In: Environment and Cancer, M.D. Anderson Hospital and Tumor Institute, pp. 354-371. Baltimore:Williams & Wilkins. 1972.

213. Van Duuren, B. L. Tumor-Promoting and Co-carcinogenic Agents inChemical Carcinogenesis. In. C. E. Searle (ed.). Chemical Carcinogens. American Chemical Society Monograph No. 173, pp. 24-51.Washington, D. C.: American Chemical Society, 1976.

214. Van Duuren, B. L., Langseth, L., Goldschmidt, B. M., and Orris, L.Carcinogenicity of Epoxides, Lactones, and Peroxy Compounds. VI.Structure and Carcinogenic Activity. J. Nati. Cancer Inst., 39: 1217-

JUNE 1978 1495



Â£.C. Miller

1228, 1967.215. Van Duuren, B. L., Sivak, A., Katz, C., Seidman, I., and Melchionne, S.

The Effect of Aging and Interval between Primary and Secondary 225.Treatment in Two-Stage Carcinogenesis on Mouse Skin. Cancer Res .35. 502-505, 1975.

216. Van Rensburg, S. J., Van der Watt. J. J., Purchase, l. F. H., Pereira 226.Coutinho, L., and Markham, R. Primary Liver Cancer Rate and AflatoxinIntake in a High Cancer Area. S. African Med. J., 48: 2508a-2508d,1974. 227.

217. Verma, A. K., and Boutwell, R. K. Vitamin A Acid (Retinoic Acid), APotent Inhibitor of 12-O-Tetradecanoylphorbol-13-acetate-induced Or- 228.nithine Decarboxylase Activity in Mouse Epidermis. Cancer Res., 37.2196-2201,1977. 229.

218. Wattenberg, L. W. Inhibition of Chemical Carcinogenesis. J. Nati.Cancer Inst., 60: 11-18, 1978.

219. Weiler, E. Die Ã„nderung der serologischen SpezifitÃ¤tvon Leberzellender Ratte wÃ¤hrendder Cancerogenese durch p-Dimethylaminoazoben- 230.zol. Z. Naturforschung., 11b: 31-38, 1956.

220. Weinstein, l. B., and Grunberger, D. Structural and Functional Changes 231.in Nucleic Acids Modified by Chemical Carcinogens. In: P. O. P. Ts'o

and J. A. DiPaolo (eds.). Chemical Carcinogenesis, Part A, pp. 217-235. New York: Marcel Dekker, 1974. 232.

221. Weinstein, l. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H.,Harvey, R. G., Harris, C., Autrup, H., Kasai, H., and Nakanishi, K.Benzo(a)pyrene Diol Epoxides as Intermediates in Nucleic Acid Binding 233.in Vitro and in Vivo. Science, 793. 592-595, 1976.

222. Weinstein, I. B., Jeffrey. A. M., Leffler, S., Pulkrabek, P., Yamasaki, H.,and Grunberger, D. Interactions between Polycyclic Aromatic Hydrocarbons and Cellular Macromolecules. In: H. V. Gelboin and P. 0. P.Ts'o (eds.), Polycyclic Hydrocarbons and Cancer: Environment. Chem- 234.

istry, Molecular and Cell Biology. New York: Academic Press, Inc., inpress.

223. Weisburger, E. K., and Weisburger, J. H. Chemistry, Carcinogenicityand Metabolism of 2-Fluorenamine and Related Compounds. Advan.Cancer Res.,5: 331-431, 1958. 235

224. Weisburger. J. H., Yamamoto, R. S., Williams, G. M., Grantham, P. H.,Matsushima, T., and Weisburger, E. K. On the Sulfate Ester of N-

Hydroxy-/V-2-fluorenylacetamide as a Key Ultimate Hepatocarcinogenin the Rat. Cancer Res., 32: 491-500, 1972.Westra. J. G., Kriek, E., and Hittenhausen. H. Identification of thePersistently Bound Form of the Carcinogen N-Acetyl-2-aminofluoreneto Rat Liver DNA in Vivo. Chem.-Biol. Interactions, 15: 149-164, 1976.Wheeler, G. P.. and Skipper, H. E. Studies with Mustards. III. In VivoFixation of C'4 from Nitrogen Mustard-C14H3in Nucleic Acid Fractionsof Animal Tissues. Arch. Biochem. Biophys., 72: 465-475, 1957.White, A., Handler, P., and Smith, E. L. Principles of Biochemistry, Ed.5, 1296 pp. New York: McGraw Hill Book Co., 1974.Wilson, R. H., DeEds. F., and Cox, A. J. The Toxicity and CarcinogenicActivity of 2-Acetaminofluorene. Cancer Res., 7: 595-608, 1941.Wislocki, P. G., Miller, E. C., Miller, J. A., McCoy, E. C., and Rosenkranz, H. S. Carcinogenic and Mutagenic Activities of Safrole, 1'-Hy-

droxysafrole, and Some Known or Possible Metabolites. Cancer Res.,37: 1883-1891. 1977.Witkin. E. M. Ultraviolet Mutagenesis and Inducible DNA Repair inEscherichia coli. Bacteriol. Rev., 40: 869-907, 1976.Wogan, G. N. Aflatoxins and Their Relationship to HepatocellularCarcinoma. In: K. Okuda and R. L. Peters (eds.), HepatocellularCarcinoma, pp. 25-41. New York: John Wiley & Sons, Inc., 1976.Wogan, G. N.. Edwards, G. S., and Newberne, P. M. Structure-ActivityRelationship in Toxicity and Carcinogenicity of Aflatoxins and Analogs.Cancer Res.,37. 1936-1942, 1971.Wood, A. W., Chang, R. L., Levin, W., Lehr, R. E., Schaefer-Ridder,M., Karle. J. M., Jerina, D. M., and Conney, A. H. Mutagenicity andCytotoxicity of Benz(a)anthracene Diol Epoxides and Tetrahydroepox-ides: Exceptional Activity of the Bay Region 1,2-Epoxides. Proc. Nati.Acad. Sei. U. S., 74: 2746-2750, 1977.Wood, A. W., Levin, W.. Chang, R. L., Lehr, R. E., Schaefer-Ridder, M.,Karle, J. M., Jerina, D. M., and Conney, A. H. Tumorigenicity of FiveDihydrodiols of Benz(a)anthracene on Mouse Skin: Exceptional Activityof Benz(a)anthracene 3,4-Dihydrodiol. Proc. Nati. Acad. Sei. U. S., 74:3176-3179, 1977.Yoshida, T Ãœberdie serienweise Verfolgung der VerÃ¤nderungenderLeber der experimentellen Hepatomerzeugung durch o-Aminoazoto-luol. Trans. Japan Pathol. Soc., 23: 636-638, 1933.




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