extremely grateful to the Burroughs Wellcome Co. and to Hoffmann-La Roche Inc. for their support of my research in Tuckahoe from 1960 to 1970 and in Nutley from 1970 to the present. Looking back at the names of earlier recipients of the Clowes Award, I realize that I am the 6th Clowes Lecturer who, at one time or another in his career, has been associated with the McArdle Laboratory for Cancer Research at the University of Wisconsin. I believe that the large number of awardees associated with McArdle is telling us something about the genius of Dr. Harold Rusch who established McArdle with a handful of outstanding young investigators. Dr. Rusch helped to develop in his associates an intense devotion to fundamental cancer research, and he provided his colleagues with an at mosphere that encouraged them to have critical but construc tive interactions with each other in ways that stimulated excel lence in research by all of the members of the group. In 1952, I had the good fortune of coming to McArdle as a graduate student of Drs. James and Elizabeth Miller. Although I failed in my first research assignment, which was to synthesize pure 2amino-1-naphthol, the Millers had great patience with me, and they tried very hard to instill their high standards into my research. Whatever small accomplishments I may have made to biomÃ©dical research were in large part the result of the excellent training and encouragement that I received from Jim and Betty Miller. An important problem that has been of great interest to me for many years is individuality in the response of human beings and other living organisms to foreign chemicals. Why does a drug or an environmental pollutant cause toxicity in one person and not in another person? Why do some cigarette smokers develop lung cancer whereas other cigarette smokers do not? One of the causes of variability in the response of human beings to a foreign chemical is individuality in the rate of metabolism of the chemical. By the mid-1960s, it was recog nized by clinical pharmacologists that the plasma half-lives and steady state plasma concentrations of drugs varied by as much as 10to 20-fold among different individuals (42, 115, 204, 318). Because it seemed likely that there were also large differences in the metabolism of chemical carcinogens in dif ferent individuals, we investigated the metabolism of BP2 by

https://cancerres.aacrjournals.org/content/canres/42/12/4875.full.pdf

Induction of Microsomal Enzymes by Foreign Chemicals and Carcinogenesis by Polycyclic Aromatic Hydrocarbons: G. H. A. Clowes Memorial Lecture

Publisher Summary This chapter provides information on the metabolism and the biological activities of polycyclic aromatic hydrocarbons. Three types of products—dihydrodiols, glutathione conjugates, and phenols—have been found as the metabolites of aromatic hydrocarbons in tissue preparations. Oxidative metabolism at the double bonds of aromatic substrates leads to the formation of epoxides. Epoxide metabolites are responsible for many of the biological effects that have been attributed to the parent hydrocarbons. The chapter discusses the role of intermediates such as free radicals, radical cations, and carbonium ions in polycyclic hydrocarbon carcinogenesis. It also discusses the factors that can influence the formation, further metabolism, and biological effectiveness of polycyclic hydrocarbon epoxides. Epoxides may prove to be biologically important derivatives that are formed from polycyclic hydrocarbons by microsomal metabolism; alternatively, they may not. The exact molecular mechanism that leads to the initiation of malignancy by hydrocarbons remains obscure, although strong suspicions persist that somatic mutations induced by metabolites are involved.

Epoxides in polycyclic aromatic hydrocarbon metabolism and carcinogenesis.

It has been demonstrated that in humans certain factors such as early menarche, late pregnancy, and nulliparity are associated with a higher risk of developing breast cancer, while early pregnancy acts as a protective factor. Induction of mammary cancer in rats by administration of the chemical carcinogen 7, 12-dimethylbenz(a)anthracene reveals that the same factors influencing human breast cancer risk also affect the susceptibility of the rat mammary gland to the chemical carcinogen. Nulliparous rats and rats undergoing pregnancy interruption are more susceptible to developing carcinomas. This fact has been attributed to the incomplete differentiation of the gland at the time of carcinogen administration. Parous rats are resistant to the carcinogenic effect of DMBA, which is explained by the complete development of the gland attained during pregnancy and lactation. This development is manifested by the differentiation of terminal end buds into secretory units, which have a smaller proliferative compartment; the epithelial cells of these secretory units have a longer cell cycle, less avidity for binding DMBA, and possess a more efficient DNA excision repair capacity.

Differentiation of the mammary gland and susceptibility to carcinogenesis

Among the multiple experimental animal models employed for analyzing the various aspects of mammary carcinogenesis, the induction of mammary tumors in rats by chemical carcinogens is one of the models most utilized. Experimentally-induced mammary tumors in rodents have proven to constitute useful tools for the study of the pathogenesis of cancer and of the molecular mechanisms involved in the initiation and progression of the neoplastic process. In vivo experimental animal models provide information not available in human populations; they are adequate for hazard identification, dose-response modeling, exposure assessment, and risk characterization, the four required steps for quantifying the estimated risk of cancer development associated with toxic chemical exposure. Using the DMBA rat mammary model, we have been able to demonstrate that the carcinogen acts on the intermediate cell of the terminal end bud (TEB), and that this structure is the one that evolves to intraductal proliferation, carcinoma in situ, and invasive carcinoma. There are several factors that regulate the susceptibility of the TEB; some of them are: a) topographic location of the mammary gland, b) age of the animal, and c) reproductive history. The understanding of the mechanisms that modulate tumorigenesis will further our knowledge and understanding in the prevention of the disease, as a result of the development of strategies for stopping the progression of the initiated cells.

Experimentally induced mammary tumors in rats.

B[a]P (benzo[a]pyrene) has been used as a prototype carcinogenic PAH since its isolation from coal tar in the 1930's. One of its diol epoxides, BPDE-2, is considered its ultimate carcinogen on the basis of its binding to DNA, mutagenicity and extreme pulmonary carcinogenicity in newborn mice. However, BPDE-1 has a similar binding to DNA and mutagenicity but it is not carcinogenic. In addition, BPDE-2 is a weak carcinogen relative to B[a]P when repeatedly applied to mouse skin, the conventional assay site. Its carcinogenicity is increased when applied once as an initiator followed repeatedly by a promoter. This indicates a major role for promotion in carcinogenesis by PAHs. Promotion itself is a 2-stage process, the second of which is selective propagation of the initiated cells. Persistent hyperplasia underlies selection by promoters. The non-carcinogenicity of BPDE-1 has yet to be resolved. PAHs have long been considered the main carcinogens of cigarette smoke but their concentration in the condensate is far too low to account by themselves for the production of skin tumors. The phenolic fraction does however have strong promotional activity when repeatedly applied to initiated mouse skin. Several constituents of cigarette smoke are co-carcinogenic when applied simultaneously with repeated applications of PAHs. Catechol is co-carcinogenic at concentrations found in the condensate. Since cigarette smoking involves protracted exposure to all the smoke constituents, co-carcinogenesis simulates its effects. Both procedures, however, indicate a major role for selection in carcinogenesis by cigarette smoke. That selection may operate on endogenous mutations as well as those induced by PAHs. There are indications that the nicotine-derived NNK which is a specific pulmonary carcinogen in animals contributes to smoking-induced lung cancer in man. Lung adenoma development by inhalation has been induced in mice by the gas phase of cigarette smoke. The role of selection has not been evaluated in either of these cases.

Synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: a bio-historical perspective with updates

K-region epoxides derived from 7, 12-dimethylbenz(a)anthracene and from 7-hydroxymethlybenz(a)anthracene were tested for their ability to induce malignant transformation of the M2 clone of fibroblasts derived from C3H mouse prostate. The parent hydrocarbons, the corresponding K-region dihydrodiols and a phenol were also tested with these cells. The K-region epoxides induced transformation but were somewhat less active than the hydrocarbons; the ring-hydroxylated derivatives were inactive. In other experiments, the addition of α-naphthoflavone was found to inhibit the formation of water-soluble metabolites from and the toxicity of 7, 12-dimethylbenz(a)anthracene without affecting malignant transformation. The results are discussed in relation to current theories regarding the metabolic activation of polycyclic hydrocarbons.



Transformation Maligne In Vitro de Fibroblastes de Souris par le 7, 12-Dimethylbenz(A)Anthracene le 7-Hydroxymethylbenz(A)Anthracene et par Leurs Derives de la Region K



L'induction de transformations malignes par les epoxydes et les dihydrodiols de la region K du 7-hydroxymethylbenz(a)anthracene et du 7, 12-dimethylbenz(a)-anthracene ainsi que par le phenol de la region K de ce dernier et les hydrocarbures dont ils sont derives, a ete eprouvee sur le clone M2 de fibroblastes issus de prostates de souris. Les epoxydes de la region K ont induit des transformations mais ont ete quelque peu moins actifs que les hydrocarbures. Le phenol et les dihydrodiols ont ete inactifs. Dans d'autres experiences, l'addition d'α-naphthoflavone a inhibe la tocixite et la formation des metabolites hydrosolubles du 7, 12-dimethylbenz(a)anthracene sans affecter la transformation maligne qu'il induit. Les resultats sont discutes a la lumiere des theories courantes sur l'activation metabolique des hydrocarbures polycycliques.

Malignant transformation in vitro of mouse fibroblasts by 7,12-dimethylbenz(A)anthracene and 7-hydroxymethylbenz(A)anthracene and by their K-region derivatives.

Induction of malignant transformation and mutagenesis by dihydrodiols derived from 7,12-dimethylbenz[a]anthracene.

Five dihydrodiols derived from benz[a]anthracene (BA) and 4 dihydrodiols derived from 7,12-dimethylbenz[a]anthracene (DMBA) have been tested, together with the parent hydrocarbons, for their abilities to induce mutations to 8-azaguanine resistance in V79 (Chinese hamster cells and malignant transformation in M2 mouse fibroblasts. The syn- and anti-isomers of benz[a]anthracene 8,9-diol 10,11-oxide were also tested for biological activity in these two systems. The non-K-region 1,2- and 3,4-dihydrodiols of BA induced mutations but the non-K-region 8,9-dihydrodiol and the K-region 5,6-dihydrodiol were inactive as mutagens; none of these BA diols transformed M2 mouse fibroblasts. The 3,4- and the 8,9-dihydrodiols derived from 7,12-dimethylbenz[a]anthracene induced mutations in V79 cells and malignant transformation in M2 mouse fibroblasts and both were more active than the hydrocarbon itself. The K-region 5,6-dihydrodiol and the non-K-region 10,11-dihydrodiol of DMBA were inactive in both test systems. The results are not inconsistent with other data suggesting that the metabolic activation of both BA and DMBA occurs through conversion of the respective 3,4-dihydrodiols into the related vicinal diol-epoxides, although other dihydrodiols may also be involved in vivo. Both the BA diol-epoxides tested were mutagenic, but although the anti-isomer transformed M2 fibroblasts, the syn-isomer was inactive.

Comparison of mutagenesis and malignant transformation by dihydrodiols from benz[a]anthracene and 7,12-dimethylbenz[a]anthracene.

Comparisons have been made between (a) the initiation of tumours in mouse skin, (b) the induction of hyperplasia and the suppression of sebaceous glands in mouse skin and (c) the induction of s.c. tumours in rats, by either benzo[a]pyrene or 7-methylbenz[a]anthracene and their related K-region and non-K-region dihydrodiols. Whilst the 3,4-dihydrodiol derived from 7-methylbenz[a]anthracene is more active than the hydrocarbon in initiating tumours in mouse skin (subsequently promoted by a phorbol ester) the 7,8-dihydrodiol of benzo[a]pyrene is very much less active than benzo[a]pyrene itself in the induction of hyperplasia or the suppression of sebaceous glands in mouse skin or in the induction of s.c. sarcomas in rats. Since much other evidence suggests that the 3,4-dihydrodiol of 7-methylbenz[a]anthracene and the 7,8-dihydrodiol of benzo[a]pyrene are the dihydrodiols involved, via the related vicinal diol-epoxides, in the metabolic activation of these hydrocarbons, mouse skin initiation-promotion experiments may be more useful for the identification of such diols than the other two in vivo tests for biological activity used here.

P L Grover

Papers

Malignant transformation in vitro of mouse fibroblasts by 7,12-dimethylbenz(A)anthracene and 7-hydroxymethylbenz(A)anthracene and by their K-region derivatives.

Induction of malignant transformation and mutagenesis by dihydrodiols derived from 7,12-dimethylbenz[a]anthracene.

Comparison of mutagenesis and malignant transformation by dihydrodiols from benz[a]anthracene and 7,12-dimethylbenz[a]anthracene.

Biological activities of dihydrodiols derived from two polycyclic hydrocarbons in rodent test systems.

Malignant transformation in vitro of mouse fibroblasts by 7,12- -dimethylbenz(a)anthracene and by their k-region derivatives.