Document 5bNLGKjvrDJMJDMdnjq3aQxYV

Reprinted from ADVANCES IN X-RAY ANALYSIS, VOL. 18 Edited bv W. L. Pickles, C. S. Barrett, J. 0. Newkirk, and C. 0. Ruud Book available from Plenum Publishing Corporation 227 West 17th Street, New York, New York 10011 ^ OX & O O O SEMI"QUANTITATIVE DETERMINATION OF ASBESTIFORM AMPHIBOLE MINERAL CONCENTRATIONS IN WESTERN LAKE SUPERIOR WATER SAMPLES P. M. Cook National Water Quality Laboratory, U. S. EPA Duluth, Minnesota 55804 ABSTRACT The amphibole mineral, cummingtonite-grunerite, has been used as a tracer for taconite tailings discharged into Western Lake Superior. The discovery of many asbestiform amphibole fibers in the tailings and Western Lake Superior water lead to concern over fiber concentrations in municipal water supplies using this water. This concern was based on the association between human asbestos exposure and increased rates of cancer of the gastrointestinal tract and peritoneum. An x-ray diffraction external standard tech nique has been developed for rapid, inexpensive, semi-quantitative determinations of amphibole mass concentration in water. The av erage amphibole mass concentrations for different Western Lake Superior water Intakes compare very well with the average electron microscope fiber counts for the same samples. Daily amphibole analysis of the Duluth water supply indicates an average amphibole concentration of 0.19 milligrams per liter. INTRODUCTION --1 O O For several year3 x-ray diffractometry has been the key an- alytical technique for National Water Quality Laboratory studies of -- the distribution and fate of taconite tailings which have been discharged into Western Lake Superior at Silver Bay, Minnesota CJ1 CO since 1956. A major component of this 67,000 ton per day discharge, the amphibole mineral cunaningtonite-grunerite, provides an ideal tracer for the tailings. The cummingtonite-grunerite (310) peak at 29.1 20 for copper Kx radiation (d 3.07 X) is not found in x-ray diffraction patterns for natural lake sediments or suspended 557 398270 STOOU4I 59 'C,8 P. M. Cook C.OANTGfTIjTOijCCeH4w7**334i -AKE 0PtN 271 fcCTERS 1 FIGURE 1----- X-RAY DIFFRACTION PATTERNS (COPPER RADIATION) FROM SEDI MENT SAMPLES TAKEN AT SUCCESSIVE 25 MM INTERVALS IN AN AREA OF TACONITE TAILINGS DEPOSITION. CUMMINGTONITE-GRUNERITE, (Mg,Fe)7Sia022(0ll)2. PEAKS ARE SHADED. THE (110) PEAK AT APPROXI MATELY 10.6 23 IS COMMON TO MOST AMPHIBOLES 398271 P. M. Cook j.')9 solids. X-ray diffraction patterns (Figure 1) of lake water sus pended solids which contain taconite tailings and sediment from successive 25 mm sections of the lake bottom in an area of tailings deposition show a clear gradation from large amounts of cummingtonite-grunerite (shaded peaks) in very recent surficial sediments to no cummingtonite-grunerite and little amphibole in the older, underlying sediments (75-100 mm). X-ray diffraction study of hundreds of river suspended sediment samples also indicates no detectable cunmingtonite-grunerite (<1Z) and only 1-22 amphibole in the nacural sediments entering Western Lake Superior. Much or all of the trace amphibole is the common, non-asbestiform mineral homblende. Further indication of the recent addition of cummingtonitegrunerite to Western Lake Superior water is provided by x-ray dif fraction patterns of many suspended sediment samples saved from the years 1940, 1950, and 1964 (Figure 2). All samples from 1940 and 1950 did not contain detectable amounts of cummingtonite-grunerite and little if any ocher amphibole minerals as indicated by a (110) peak at 10.6 29 (d = 8.34 A). All of Che 1964 samples, however, contained large concentrations of cummingtonite-grunerite as shown by che appearance of large (110) and (310) peaks. The (110)/(310) peak ratios for these samples are typical of those found for tac onite tailings samples. s T004 4 160 FIGURE 2----- X-RAY DIFFRACTION PATTERNS FOR SUSPENDED SOLID SAMPLES OBTAINED FROM THE DULUTH MUNICIPAL WATER SUPPLY INTAKE: A HISTORI CAL RECORD OF AMPHIBOLE CONCENTRATIONS IN DULUTH'S DRINKING WATER 398272 v '.60 P. M. Cook In 1973, study of the morphology of amphibole particles in fine taconite tailings bv transmission electron microscopy revealed the presence of many as'.astiform fibers (Figure 3). The realization that many of these cummingtonite-grunerite fibers are indistin guishable from amosite asbescos fibers lead to concern over the use of Western Lake Superior water for municipal drinking water supplies. This concern was based on the association between human asbestos exposure and increased races of cancer of Che gastro intestinal tract and peritoneum (1) and daily x-ray diffraction analyses of Duluth, Minnesota drinking water samples which indicated che constant presence of high, concentrations of taconite tailings. Transmission electron microscope analysis of Duluth water samples confirmed the presence of many amphibole fibers. Since che discovery of asbestiform amphibole fibers in the water supplies of Silver Bay, Beaver Bay, Two Harbors, Duluth, and Cloquet, Minnesota, extensive sampling programs by the Environmental Protection Agency and other groups have been undertaken for electron microscope fiber counts. These analyses while in agreement with che x-ray diffraction results, are very expensive, time-consuming, and imprecise. At this time fiber counts done by different lab oratories are nor comparable and intralaboratory replicate results usually vary by 50% of the mean. The amphibole fiber concen trations generally correlate with the amphibole mass concentrations determined by x-ray diffraction. Thus x-ray diffraction monitoring of water samples combined with occasional electron microscope fiber counts offers a faster, less expensive, and probably more accurate measure of amphibole fiber contamination. This technique has been particularly useful for evaluating various filtration media's abil ities to remove amphibole fibers from drinking water. AMPHIBOLE ANALYSIS OF WATER SAMPLES Water samples from Western Lake Superior public water supplies, normally ten liters in volume, are pressure filtered through 0.45u membrane fibers. When the turbidity of the sample is known, the volume filtered is adjusted to give a 4-8 mg sediment sample. The total suspended solids are determined by difference and a weighing correction applied to compensate for a small filter weight loss due to leaching (2). Distilled water blanks are run periodically to check for contamination. The dry membrane filter with sample is fastened to a glass slide with a thin film of lacquer, the filter edges trimmed, and the slide directly examined with a Norelco ver tical diffractometer (copper radiation) with a graphite crystal focusing monochromator. The amphibole fibers and cleavage fragments assume a preferred orientation such that the c-axis, which corresponds to the long dimension of the fiber, is parallel to the filter surface. This STOOU U I 6I 398273 P. M. Cook 561 29/1'lOOiS FIGURE 3------ELECTRON MICROGRAPH OF <2u TACONITE TAILINGS. a) LOW MAGNIFICATION (2,500X). b) HIGHER MAGNIFICATION (12.500X) VIEW OF AN AMPHIBOLE FIBER BUNDLE causes the (110) reflection and, to a lesser extent, the (310) reflection intensities to be enhanced, permitting the detection of trace amounts of amphibole. As little as 0.05 mg of <2u cusiningtonite-grunerite produces measurable (110) and (310) peaks. A semi-quantitative measurement of the amphibole concentration is made by an external standard technique. This technique has been 398274 562 P. M. Cook used to estimate trace amounts of chrysotile asbestos and amphibole asbestos in dust samples on membrane filters (3,4) and fulfills the need for rapid, standardized estimates of amphibole concentration in samples which are not amenable to the use of an internal stan dard. Three potentially large sources of systematic error had to be considered before accepting the external standard model; varia bility of particle size, sample mass absorption coefficient, and amphibole preferred orientation. The external standard chosen for the preparation of standard curves of x-ray peak intensity versus mass of amphibole was the amphibole mixture found in the <2y taconite tailings. This choice was made since the predominant amphibole in Western Lake Superior water is cummingtonite-grunerite from taconite tailings and natural amphibole concentrations in Lake Superior water are normally not detectable by x-ray diffraction. The <2u taconite tailings were determined by the x-ray diffraction of cummingtonite-grunerite/ quartz mixtures to contain approximately 802 amphibole and 202 quartz. Most of the amphibole is cummingtonite-grunerite with some actinolite-tremolite. Larger size fractions of the tailings contain less amphibole and more quartz with a small percentage of magnetite. Reference samples were prepared by adding known amounts of the <2u amphibole standard to ten liter samples of Lake Superior water having no detectable amphibole minerals. This water, obtained from Grand Marais, Minnesota, contained 0.4 mg/1 suspended solids which consisted primarily of organic debris, diatoms, quartz, and clay minerals. These standard samples were then filtered and analyzed by x-ray diffraction in the same manner as unknown samples. The re sulting x-ray diffraction patterns are identical in appearance to those for Duluth water samples. The <2y amphibole particle size Cby gravity settling) for the external standard was shown to be appropriate by a centrifugation sizeseparation of Duluth water suspended solids from samples taken on fifteen different days. Ninety-five percent of the suspended solids were in the <2u fraction with only a small amount of amphibole in the 5% which was >2u. Thus variability in diffracted x-ray inten sity due to mineral particle size >2u is insignificant. The filtration of ten liters of Duluth water normally results in 4-8 mg of suspended solids retained on the 0.45u membrane filter. When the suspended solids exceed 0.8 mg/1, smaller volumes are filtered. A sample weight of 8 mg and an average density of 2 g/cm3 results in a hypothetical sample thickness of 3u on the filter. This -thin sample thickness should preclude variability due to dif ferences in sample absorption coefficients. Direct evidence for this is provided by the linearity of a plot of percent amphibole versus x-ray intensity for samples in this weight range; the uniform ' STOOUUl63 398275 P. M. Cook 563 intensity of filter background in the x-ray diffraction pattern with increasing sample weight to 10 mg; and the linearity of a plot of quartz peak (d = 3.33 k) intensity versus weight of quartz, regard less of total sample weight in the range 0-12 mg. 3<XV AMPHIBOLE (110) PEAK X-RAY INTENSITY counrs/second FIGURE 4----- EXTERNAL STANDARD CURVE FOR AMPHIBOLE SEMI-QUANTITATIVE ANALYSIS The non-linearity of the external standard curve (Figure 4) is due to a decreasing degree of preferred orientation as the amount of amphibole increases. This is indicated by decreasing amphibole (11Q)/(31Q) and amphibole Q.10)/quartz peak ratios with increasing weight of the standard amphibole-quartz mixture on the filter. The utility of the external standard curve depends on how well the curve models amphibole preferred orientation in environmental samples. Similar curves based on samples prepared with Increased amounts of natural sediment agreed well with the standard curve used. With large amounts of natural sediment, the amphibole peak intensity is we.akened which would cause an underestimation of amphibole concen trations. Other standard curves were employed to estimate the amphibole concentration in the few samples with a very high concentration of non-amphibole minerals. 4ST004 164 398276 :>64 P. M. Cook External standard curves, such as Figure 4, were plotted from the non-linear least squares refinement of amphibole mass versus amphibole (110) peak intensity data points. The data fit an equa tion of the form: L-. IQ + I(l - exp-kC), where I; = intensity at concentration C (mg amphibole); I0 = intensity at C = 0; ls = intensity at C = =; and k is a constant. This equation is consist ent with a model in which tne degree of preferred orientation decreases as more amphibole particles are placed on the membrane filter. Standard curves utilizing amphibole (110) peak height above background are identical to curves plotted from the (110) peak areas. Both measurements are used and give the same amphibole concentrations for environmental samples. Use of an amphibole (310) peak curve gives the same results but with less precision due to lower peak intensity. Replicate (five) analyses of Duluth water samples indicate a standard deviation of + 3% for determining amphibole concentrations in typical samples with 0.1-0.3 mg/1 amphibole. For samples having lower amphibole concentrations (<0.1 mg/1) and high suspended solids (>1.0 mg/1), this precision is reduced to + 25%. Overall suspended solids determinations have a standard deviation of + 6X of the mean. Detection limits for determining amphibole concentration depend on the volume of water filtered and can be as low as 0.5 ug/1. WATER SUPPLY AMPHIBOLE ANALYSIS Daily analyses of Duluth water samples for amphibole and sus pended solids concentrations began in March 1973 and continues to date. Results through January of 1974 are shown in Figure 5 with climatological data and intake water temperatures. X-ray diffrac tion analysis provides a picture of daily and seasonal fluctuations in amphibole and suspended solids concentrations. For example, periods of heavy rainfall are followed by abrupt increases in suspended solids due to river run-off and shore erosion. These increases in suspended solids do noc coincide with increases in amphibole, indicating a different source for amphibole sediment. Maximum amphibole concentrations (up to 0.8 mg/1) occur in the lace fall and spring. Minimum amphibole concentrations (0.04 mg/1) occur during the late summer and early fall when a thermocline is present in Western Lake Superior. The average amphibole concentration measured was 0.19 milligrams per liter with 0.83 milligrams per liter total suspended solids. -During the period August 22-November 28, 1973, personnel from Region V of the Environmental Protection Agency obtained weekly water samples from municipal water supplies using Lake Superior water from Grand Marais, Minnesota to Marquette, Michigan. These 398277 s g m o o is P. M. Cook 363 c o in o 3. j9)0m a*o"i pu M UIP jOUO)|dOOJd I /fi'JJ (pilot papuadsns -=>j S Oaas, CO z *Ho"H CadiJ 3P 2fZCU--sJ <PrUaLd. oCJ wH CO W a id ^OJ <t2-* CO I~t QW CUd a2 HC a^4 3 co a a2 CO < Q< 35 aj Q -j a o< --ihCHQ CJ i XCU yO 5cS > < -j x cQI II (/> CD. CD. C* e' er* cn .',66 P. M. Cook samples were analyzed for amphibole mass concentration at the Nacional Water Quality Laboratory and amphibole fiber concentration by transmission electron microscopy at the Ontario Research Foundation in Sheridan Park, Ontario and McCrone Associates in Chicago, Illinois. Figure 6 depicts the average x-ray diffraction and electron microscope measurements for each station. The agree ment between these two measurements is obviously very good. The pattern of maximum concentrations at Beaver Bay and decreasing concentrations in a counterclockwise direction around Western Lake Superior is consistent with large quantities of amphibole fiber discharged at a point between the Silver Bay and Beaver Bay, Minnesota water supply intakes and then transported towards Duluth (southwest) by the predominantly counterclockwise currents of Western Lake Superior (5). Comparison of NWQL X'Ray Diffraction Amphibole Analyses to EPA, Region V Electron Microscope Fiber Counts for Public Water Supply Samples- Average Concentrations for Weekly Samples Taken Aug 22 - Nov 28, 1973 0 40r >--1; m$ 1I in j I i mI FIGURE 6--COMPARISON OF AMPHIBOLE MASS CONCENTRATION DETERMINED by x-ray diffraction to transmission electron microscope amphibole FIBER COUNTS FOR LAKE SUPERIOR WATER INTAKES L3 I *1*10015 398279 P. M. Cook ">67 ACKNOWLEDGEMENTS The author wishes to gratefully acknowledge the assistance of Mr. lames Tucker of the National Water Quality Laboratory for electron microscope examinations of water and tailings samples; Mr. Robert Fulton and Mr. David Marklund for their excellent work in preparing many of the samples examined by x-ray diffraction; and Dr. 3illy Fairless of the Environmental Protection Agency, Region V, Central Regional Laboratory, for providing water intake fiber counts. REFERENCES 1. I. J. Selikoff, E. C. Hammond and J. Churg, "Carcinogenicity of Amosite Asbestos," Arch. Environ. Health 15, 183-186 (1972). 2. J. G. Eaton and G. E. Likens, "Use of Membrane Filters in Gravimetric Analyses of Particulate Matter in Natural Waters," Water Resources Res. 1151-1156 (1969). 3. A. L. Rickards, "Estimation of Trace Amounts of Chrysotile Asbestos by X-Ray Diffraction," Anal. Chem. 4, 1872-1873 (1972). 4. J. V. Crable, "Quantitative Determination of Chrysotile, Amosite, and Crocidolite by X-Ray Diffraction," Am. Ind. Hyg. Assoc. J. 27, 293-298 (1966). 5. C. E. Adams, "Summer Circulation in Western Lake Superior," Proc. 13th Conf. on Great Lakes Res., 862-879 (1970). STOOH 168 398280 n Cancer Research Volume 30 [CANCER RESEARCH 30. JS9-576, March 19701 MARCH 1970 Number 3 Carcinogenesis by Chemicals: An Overview--G. H. A. Clowes Memorial Lecture1 ( v | JaImt eiss aA.treMmeilnledrous honor to be chosen as the Ninth past decade, forms the greater pan of this talk. Both of G. H. A. Clowes Memorial Lecturer and it is one that I us owe a great deal to our many collaborators over the MshcAarredlefuLlalybowraittohrymfoyr cCoanwcoerrkReerseDarcr.h. EUlinzioaebresttthy oCfaWviescrot nMsinillMeerd.ical yCeeanrtesr,. Manaddisonno, thWinisgconcsainn53c7o06mpare with the steadfast sup I never cease to marvel at my good fortune in having port and encouragement that we have received from Dr. her as a marital and research partner since our graduate Harold P. Rusch, Director and indeed the builder: of the student days. Our work together, particularly that of the McArdle Laboratory since its inception in 1940. We know that our colleagues at McArdle join us in voicing our great esteem for Harold Rusch. Dr. G. H. A. Clowes helped to found this Association in 1907 and was for many years Director of Research of Eli Lilly and Company. Clowes' essay in 1956 on "Cancer Research Fifty Years Ago and Now" (20) provides in teresting perspective for these days of relative freedom and affluence in research. Clowes wrote: "In comparing conditions 50 years ago with those of the present, the greatest contrast is to be found in the freedom now granted to research workers in choosing a field of investi gation and deciding what course to follow." "... whereas 50 years ago there was little or no incentive to engage in cancer research, today the results being obtained in this field are so important and the opportunities for re search are so excellent that able young investigators need have no hesitation about engaging even in long-term projects in this most difficult and at the same time most fascinating field." Dr. Clowes was intensely interested in understanding basic aspects of many facets of metabolism and growth of normal and malignant cells. This talk in his remem brance is directed in a modest way toward similar goals. CARCINOGENESIS IN MAN AND EXPERIMENTAL ANIMALS 3 r i. oo --i CD CD cn co ' Presented at the 60th Annual Meeting of the American Associa tion for Cancer Research. March 1969, San Francisco. Calif. The work of the author and his associates has been supported by Grants CA-0717J *nd CRTY-5002 from the National Cancer Institute, USPHS. and by iranu from the Jane Coffin Childs Memorial Fund for Medical Re tard! and the Alexander and Margaret Stewart Trust Fund. As oncologists all of us seek to understand and control neoplasia in our species, and it is evident that the degree of understanding and control that any scientific genera tion achieves depends largely on the concepts and tools it can devise. Looking back, one sees that among the March 1970 398281 559 James A. Miller major contributions of research in oncology in the latter haIf of the 19th century were the recognition and descrip tion of the great variety of malignant and benign neo plasms in man and other species as somatic cellular dis eases. Likewise, among the successes of cancer research in this century stand the discoveries of the wide variety and large number of chemicals, viruses, and radiations that can induce cancer in mammalian species. Certainly, everyone would like to see cancer research in the re maining decades of this century reveal the molecular na tures of carcinogenic processes and the molecular pheno types of the neoplasms these processes produce. One cannot help but feel that even partial successes in these difficult tasks will spark major advances in the therapy and prophylaxis of cancer in the human. So let us hope that this Association will be out of business by the year 2000! Any overview of chemical carcinogenesis should start with comments on the observations of the London surgeon Percivall Pott (125) on the occurrence of scrotal skin can cer in English chimney sweeps. Now almost 200 years old, Pott's implication of gross contact with soot as a cause of this neoplasm and his emphasis on its long latent period of development stand as milestones in oncology. Pott did not suggest avoidance of contact with soot as a means of prevention. Perhaps he thought it impractical for a chimney sweep--an essential worker in Pott's time. Still, as Clemmesen (19) has noted, Pott's book in 1775 with its brief paragraphs on the "soot wart" apparently inspired a ruling 3 years later by the Danish Chimney Sweepers' Guild that its members should bathe daily. The value of that ruling was revealed a century later when Butlin (16) investigated the relative rarity of scrotal skin cancer in chimney sweeps outside of England and found that on the continent it was apparently pre vented by frequent bathing and protective clothing. How simple this seems today when we make only painfully slow progress toward the elimination of a personal form of air pollution that has led to an epidemic of lung cancer in the human and is tenaciously fostered by habit and the cigarette industry. From Pott's time to our present chemical age, preven tion of contact with harmful chemicals is a lesson that has had to be taught again and again. Violations of this Tabic i Chemicals recognized as carcinogens in the human Agem Target Certain soon. tars, oils (18) Cigarette smoke (14b) 2-Naphthvlamine (18) 4-Aminobiphenvl (18. 77) Benzidine (18) /V./V-Bis(2-chloroethyl> 2-naphihylamine (155. 160) Bis(2-chloroeihyl)sulfide (162) Nickel compounds (18. 152) Chromium compounds (18) Asbestos (18. 163) Skin, lungs JLungs Urinary bladder Urinary bladder Urinary bladder Urinary bladder Lungs Lungs, nasal sinuses Lungs Lungs, pleura Table 2 Some eariv landmarks in experimental chemical carcinogenesis 1915 Induction of skin cancer in rabbits 1918 and mice by coal tar 1930 1933 1933 1936 1937 1941 Tumor induction by the first pure chemical carcinogen-1.2.5.6- di benzanthracene Isolation of the carcinogen 3,4benzpyrene from coal tar Inducuon of liver cancer in rats by o-aminoazotoluene and by p-dimethylaminoazobenzene Induction of urinary bladder cancer in dogs by 2-naph(hylamine Initiation and promotion suges in skin carcinogenesis with tar and 3,4-benzpyrene Yamagiwa and Ichikawa (173. 174), Tsutsui (158) Kennaway and Hieger (71) Cook. Hewetu and Hieger (23) Yoshida (175), Kinosita (75) Hueper, Wiley, and Wolfe (60. 61) Bercnblum (4, 5); Rout. MacKenzie, and Kidd (93. 135) simple but difficultly appreciated and applied rule have led repeatedly to the discovery of a variety of chemical carcinogens with man as the unwitting species at risk. These carcinogens are listed in Table 1. One can only ad mire the efforts of the epidemiologists who established these cause and effect relationships from thousands of human tragedies. Experimental confirmation of the first recognized chemical carcinogens for the human was sought quite early, but the use of limited dosages and times of ex posure coupled with unfortunate choices of species led to many failures (59, 149). The first breakthrough was re ported from Japan in 1915, and a number of important landmarks in experimental chemical carcinogenesis oc curred in the subsequent 3 decades (Table 2). The preoc cupation with aromatic carcinogens then gave way to the discovery of a wide variety of aliphatic carcinogens and some carcinogenic metals. From the beginning, the chem ical carcinogens have provided tools for studies on mecha nisms of action of carcinogenesis. The discovery of new chemical carcinogens continues unabated and still oc casionally derives from exposures of humans to various compounds. Concomitant with these discoveries of chem ical carcinogens, a wide variety and large number of carcinogenic or oncogenic viruses (14, 50, 161) have been revealed. Likewise, X-rays and ultraviolet light, early shown to be carcinogenic in man and then in experimental animals, have been joined by a variety of carcinogenic ionizing particulate radiations (6, 55, 130). So today we know that we are confronted with a host of carcinogenic chemicals, viruses, radiations, and complex interplays between these agents as models and examples of causes of cancer in man. It seems certain that many new chemical and viral carcinogens will be found in fu ture work. Recent work of the geographical pathologists (57, 121) has raised strong suspicion that a high propor tion of human cancer is of environmental origin. The chemical carcinogens in our total environment, through lifetimes of exposure to small amounts of these com pounds, may rank as major causes of human cancer. . This in no way minimizes the possible role of viruses in carcinogenesis in the human. Present knowledge on the 560 CANCER RESEARCH VOL. - J*UIJ 398282 ^ Clowes Memorial Lecture oncogenic viruses makes it almost inconceivable that our species could be exempt from such causes of cancer. It seems far more likely that the important question will be the degree to which such agents act alone, in concert with, or following the action of chemical and physical carcinogens in the human. If, for example, some or many chemical carcinogens activate or act with oncogenic viruses, the removal of the chemical carcinogen might frequently be the most feasible course of action. The rec ognition and control of chemical, viral, and physical car cinogens in man's external and internal environment has become a principal task of cancer research. PYRROLIZIDINE ALKALOIDS (SENECIO, CROTOLARIA AND HELIOTROPIUM GENERA) AFLATOXIN B, (ASPERGILLUS FLAVUS STRAIN) "\ .0 - GLUCOSYL-O-C^-NssN-CHj 0 ch2-ch=ch2 CHEMICALS AS CARCINOGENS Chemical carcinogens now comprise a very diverse group of nonviral and nonradioactive organic and inor ganic structures with various species and tissue selectivities. Some of these agents have always been present in inanimate nature, many of them are products of man's day to day activities, still more of them derive from man's great synthetic capabilities in the laboratory, and some of 7,12-DIMETHYLBENZto (ANTHRACENE ( ,10 - DIMETHYL-1,2-8*1 /C0CH3 N 'h 2- ACETYLAM1N0FLU0RENE CH3 N-NO CH3 N-METHYL-4-AMIN0AZ0BENZENE DIMETHYLNITROSAMINE CjHj- S-CHj-CHj-CH - COOH NH2 ETHIOMINE I 0 4-NITROOUINOUNE-l-OXIDE ca4 CARBON TETRACHLORIDE vy*s*NH2 0 ETHYL CARBAMATE (URETHAN) Chart l. Some lynthetic chemical carcinogens for experimental ani mals (18). 1 The abbreviations used are; AAF, 2-acetylaminofluorene: MAB, N-meihyW-aniinoazobenzene-. AF. 2-aminofluorene. CYCASIN (CYCAD NUTS) SAFROLE (OIL OF SASSAFRAS) Chart 2. Some naturally occurring compounds which are carcino genic Tor experimental animals (110. 112). the newest chemical carcinogens are metabolites of liv ing cells. Some of the variety to be found among the synthetic chemical carcinogens is shown in Chart l. The very po tent hydrocarbon 9,10-dimethyl-1,2-benzanthracene, the versatile aromatic amide AAF,2 the aminoazo dye Nmethyl-4-aminoazobenzene, and the nitrosamine dimethylnitrosamine are each representative of large groups of similar compounds with various species and tissue spec ificities. The heterocyclic compound 4-nitroquinoline-loxide and its close relatives were discovered to be car cinogenic relatively recently and have carcinogenicities similar to those of the polycyclic aromatic hydrocarbons. Ethionine, an amino acid which is hepatocarcinogenic in the rat, was originally designed as an antagonist of methi onine. The mouse and hamster hepatocarcinogen carbon tetrachloride and the versatile water-soluble carcinogen ethyl carbamate further exemplify the variety of struc tures found among chemical carcinogens. The variety of carcinogenic structures seems almost endless, and some of the newest and most exotic struc tures have been found as metabolites of fungi and green plants (110, 112). Some of them, such as the aflatoxins, the pyrrolizidine alkaloids, and cycasin (Chart 2), have very high hepatocarcinogenicities in the rat. The aflatox ins are suspected as being among the causes of the high incidences of primary hepatic cancer in native popula tion groups in central and southern Africa and else where (2, 7, 76, 170). An important aspect of carcinogenesis by the synthetic and naturally occurring carcinogens shown in Charts 1 and 2 is that studies on their mechanisms of action indi cate strongly that most if not all of these compounds are not carcinogenic as such. They appear instead to be precarcinogens which are converted in the host into carcir.ogenic and reactive structures. In this respect, they differ from the carcinogenic alkylating agents shown in Chart 3. These agents are essentially in their Anal reactive forms as administered and they generally take part in S*2 (substitution, nucleophilic, bimolecular) reactions in I L I *1*1001 S march 1970 ijrjy 398283 561 James A. Miller 0 ,CH,-CHT7CI r: . j* |] ch2- H URACIL MUSTARD l-ETHYLENEOXY3,4-EPOXYCYCLOHEXANE :>ch2 CHjtCH^jgCO-N | xCH2 N-STEAROYLETHYLENE IMINE ch2-- ch2 0 * fs-PROPIOLACTONE 9 ...9 CHj-S-O-^C^T-O-S-C^ 06 (,4 -01 METHANESULFONQXY* BUTANE ch2 /\ CH, CH* A' / -o--1-0 0 PROPANESULTONE Chart 3. Some alkylating agents which are carcinogenic for expertmental animals. Dashed lines, bonds which are cleaved in the alkyla tion reactions. which a relatively positive or electrophilic atom in the alkylating agent combines with relatively negative or nudeophilic atoms of the molecules attacked in cells (129, 134, 141). During administration to tissues, these carcino genic electrophilic reactants encounter extracellular nu cleophiles such as water and protein before their entry into cells. This may account in part for the observations that the alkylating agents are frequently not strong car cinogens and may require multiple large dosages at local tissue sites to reveal their activity as carcinogens. This is not always true, however, for the uracil mustard shown in Chart 3 is a potent lung carrinogen in the mouse (1). Similarly, propanesultone, one of the newest cardnogenic alkylating agents (30), is a potent cardnogen in the sub cutaneous tissue of the rat and even yields some tumors in distant neural tissue in this spedes. The small class of cardnogenic metals (18, 43) such as beryllium, cadmium, cobalt, nickel, and lead is another group of agents which, in their ionic forms, are electro philic reactants and can react with various nucleophiles. Organic electrophilic derivatives of metals may also be formed in vivo. Carcinogenesis by metals, espedally with regard to mechanisms of action, has been a relatively ne glected area. Studies on nickel carcinogenesis (43, 132) are an important exception, and similar investigations should aid in an understanding of cardnogenesis by these seemingly simple chemicals. MECHANISMS OF ACTION OF CHEMICAL CAR CINOGENS: GENERAL ASPECTS Since it seems axiomatic that cardnogenic agents must induce neoplasia through interaction with tissue compo- nents. efforts to understand the mechanisms involved in chemical cardnogenesis have been directed primarily to. ward studies of the chemical reactivities of the cardno- gens and their metabolites. Thus, the determination of the active form(s) (ultimate cardnogens) of a chemical cardnogen comprises a logical first step in the elucidation of it$ mechanism of action. Most of the current work in chemical cardnogenesis is now focused in this area and at the next step, i.e., the eluddation of the nature of the inter actions of these active form(s) with tissue constituents, -S3 These studies are increasingly providing possible mechanisms by which chemical cardnogens may produce tu mors, but the really difficult problems are still ahead. That is, we must determine which of these interactions are critical to particular cardnogenic processes and the mechanism(s) by which these critical interactions induce neoplasia. These are formidable problems which must still be met not only in the study of chemical cardnogene sis, but in the eluddation of the mechanisms involved in viral and radiation cardnogenesis as well. So far, it has not been possible to guide these determinations from a knowledge of the biochemical nature of the neoplasms produced by these agents, for the neoplastic properties of no tumor are understandable as yet in terms of its molec ular phenotype. These problems in cardnogenesis at the molecular and cellular levels have been discussed in detail recently in an excellent review by Farber (38). Macromokcule-bound Derivatives of Chemical Cartinogens in Vivo. The first covalent interactions of chemical cardnogens with proteins of target tissues were noted over 20 years ago (105, 106), and the earliest studies on the re action of nucleic adds with alkylating agents in oivo were made in 1957 (167). Covalent bindings of residues of chemical cardnogens with macromolecules (DNA. RNA, and/or proteins) in oivo have now been noted in all cases which have been adequately examined (38, 107, 115). T These studies have most frequently involved examinations * of the liver or skin of treated animals, but macromolecules Jj from a wide variety of other tissues have been studied after 1 treatment with spedfic chemicals. Likewise, these studies have utilized a wide variety of chemical cardnogens: the ^ aromatic amines, aromatic polycyclic hydrocarbons, alkyl- J ating agents, and potential alkylating agents. Interactions j of derivatives of these and other chemical cardnogens with r other macromolecules [e.g., glycogen (35, 38)] and with lower-molecular-weight compounds of tissues surely also; occur. However, the protein and nucleic add interaction] have been the focus of interest since, in our present state of knowledge, we look chiefly to the proteins and nucleic* adds for those changes which could explain the heritable f and at least quasipermanent partial or complete loss growth controls that characterizes neoplasms. Indeed, al number of correlations have been obtained in which the?* amount of 1 or more of these covalent bindings appears 0 ' correlate with the ultimate inddence of tumors (12, 106, 107). However, these correlations with binding * * total DNA, RNA, or protein are not exact and need cc siderable refinement before far-reaching assessments their roles in the cardnogenic process can be made. 562 CANCER RESEARCH VOL-3%j UHl/iOOlS Tf. iu. -.11 ia.J*U 398284 Clowes Memorial Lecture ^ r=\ (T\ r)/C0CH} \N // \ --/ ~u j.ACETYLAMINOFLUORENEIAAF) - CARCMOGEMC N UVER ANO/OR OTHER TISSUES OR RAT, MOUSE, HAMSTER, RABBIT, 000,CAT, FOWL A homooehate NADPH UVER ENDOPLASMIC RETICULUM + NADPH Oj NOT ACTIVE IN GUINEA PIO N-HYDROXY-AAF MORE CARCINOQENIC THAN AAF , ESPECIALLY AT SITES OF APPLICATION IN RAT, MOUSE,HAMSTER, RABBIT, ANO GUINEA PIG NUCLEIC ACID- ANO PROTEIN-BOUND DERIVATIVES Chart 4. The primary activation of the carcinogen AAF by /Vhydroxylation (25, 109). Reactive Forms of Chemical Carcinogens. Although the studies on the covalently bound forms of chemical carcin ogens in cells have not revealed the molecular aspects of carcinogenesis that we seek, these studies have provided considerable insight into the reactive forms of chemical carcinogens in vivo. As noted later in this talk, an impor tant product of this work in the past few years has been the realization that, despite their chemical dissimilarities, many chemical carcinogens are metabolized in vivo to potent electrophilic reactants. The properties of these re active forms of the carcinogenic aromatic amines and amides are considered next in some detail, since they form a well-documented example. noncarcinogenic metabolite of AAF (58, 118), converts it into a potent carcinogen for the rat (53). On the other hand, the introduction of a Af-hydroxy group into an aro matic amine or amide is not necessarily sufficient to con vert it into a carcinogen; as shown in both our studies (104, 136) and those of Gutmann et al. (53, 54), the aryl group is a very important determinant. The greater carcinogenic activity of the metabolite /V-hydroxy-AAF, as compared to that of the parent amide (109), and the higher yields of macromolecule-bound fluorene derivatives (Chart 4) in the livers of rats given /V-hydroxy-AAF rather than AAF (27, 63, 97), implicate iY-hydroxy-AAF as an intermediate in the carcinogenicity and in vivo reactivity of AAF. However, the fact that A^-hydroxy-AAF, like AAF, has very little ability to react under physiological conditions with proteins or nucleic acids or their derivatives (3, 48, 64, 73, 102, 103) indicates that some further metabolic step(s) are necessary to con vert A-hydroxy-AAF to ultimate carcinogenic and reac tive forms. Since our first evidence that this metabolic ac tivation depended on esterification of the TV-hydroxy group to yield strong electrophilic reactants came from studies on MAB, a hepatocarcinogenic aminoazo dye, these data will be considered first. Metabolic Activation of MAB in Vivo. The first hints as to the nature of the reactive forms of MAB in vivo came from studies with Drs. Scribner, Poirier, and Lotlikar. The first step was the finding that treatment with cold alkali of the liver proteins from rats fed MAB yielded CARCINOGENESIS BY AROMATIC AMINES AND AMIDES A-Hydroxylation of Aromatic Amines and Amides. The propensity of the aromatic amine and amide carcinogens to induce tumors in tissues distant from the sites of entry, but not at these sites, in man and experimental animals has been the basis for many years of the idea that these compounds require metabolic activation. Evidence that (V-hydroxylation is the initial activation step for the car cinogen AAF was first obtained in our laboratory in 1960 with Dr. John Cramer (25) (Chart 4), and this conclusion is now supported by studies from several laboratories with a variety of carcinogenic aromatic amines and amides (113, 114). In some cases, the inability of the animal to iV-hydroxylate a sufficient amount of an amide appears to be the crucial factor which prevents an amide from being a carcinogen. For instance, the guinea pig has little or no ability to jV-hydroxylate AAF; AAF is not carcinogenic for this species while JV-hydroxy-AAF does produce tu mors (108). Similarly, Gutmann el al. (53) have shown that the synthetic iV-hydroxylation of 7-hydroxy-AAF, a Chart 5. The nonenzymatic reactions of the synthetic ester ,Ybcnzoyioxy-MAB with certain amino acids at neutral pH and the iden tity of some of these in oitro products with derivatives released on hy drolysis of the liver protein from rats fed MAB (88, 89. 92, 124. 137). March 1970 563 398285 STOCK,l, | 73 * l James A. Miller 3-methylmercapto-MAB, in which the thiomethyl group iV-hydroxy-AAF with the hepatocarcinogenidty of this was derived from methionine (114, 137) (Chart 5). Shortly hydroxamic add (see below). afterward, /V-benzoyioxy-MAB. synthesized as a possible Metabolic Activation of /V-Hydroxy-AAF in Ww. The precursor of /V-hydroxy-MAB (124). was shown to be a initial studies on the reactivity of JV-benzoyioxy-MAB led potent electrophilic reactant. It reacts with methionine quickly to similar studies with synthetic esters of N-by- nonenzymatically at pH 7 to yield a water-soluble deriva droxy-AAF with Drs. Lotlikar, Scribner, and DeBaun. tive which decomposes to 3-meihylmercapto-MAB (92, Like iV-benzoyloxy-MAB, TV-acetoxy-AAF and A'-benzoyl-J 124). These observations led to the suggestion that the oxy-AAF are potent electrophilic reactants. They react, metabolically active form of MAB in the rat liver is an with methionine and methionyl peptides, and the resulting ester of jV-hydroxy-MAB. sulfonium derivatives decompose to yield a mixture of j Further evidence that a reactive form of MAB in oioo 1- and 3-methylmercapto-AAF (27, 92, 103) (Chart 6). The i might be an ester of AMiydroxy-MAB has come from the liver proteins from rats given AAF or A'-hydroxy-AAF! studies with Dr. Lin (88, 89) on the characterization of yield these same o-methylmercapto-AAFs on treatment j the protein-bound dyes in the livers of rats fed MAB. The with cold alkali (27). Degradation of the peptide-bound J polar dyes released by hydrolysis of the liver proteins can sulfonium derivatives can occur by an internal nucle also be formed by reaction of W-benzoyloxy-MAB with ophilic attack which causes cleavage of the peptide linkage] the appropriate amino add (Chart 5). The major polar (Chart 6) in a manner analogous to that reported by Gross] dye released by this procedure is 3-(homocystein-5-yl)- and Witkop (49) and Gundlach et at. (52) for alkylated] MAB; this dye presumably arises in dido through the methionine residues in peptides. To the extent that this] 5-demethylation of protein-bound 3-(methion-i'-yI)-MAB. type of decomposition occurs in oioo, the fluorene residues] It can be formed nonenzymatically by reaction of /V-ben- would be detached, the protein would be cleaved at the] zoyloxy-MAB with homocysteine. Two other polar dyes former methionyl peptide linkage, and one of the resulting] derived from the hepatic protein-bound dyes are identical peptides would contain homoserine instead of methionine.! with 3-(3-tyrosyl)-MAB and yV-(3-tyrosyl)-MAB synthe The protein-bound derivatives in the rat liver which gives sized by reaction of yV-benzoyloxy-MAB with tyrosine. rise to 1- and 3-methylmercapto-AAF account for only > iV-Benzoyloxy-MAB also reacts with tryptophan and cys about 15% of the total protein-bound fluorenyl derivative teine, but no polar dyes containing these amino adds were By analogy with the protein-bound forms of MAB in rat* detected. The lack of detection of a cysteinyl polar dye is liver and from the known reactivity of esters of TV-hydroxy-* in accord with the extreme lability of the cysteinyl deriva AAF, fluorene derivatives of homocysteine, cysteine, tyro-^ tive prepared by reaction with /V-benzoyloxy-MAB to the sine, and tryptophan may also occur in the livers of rats'* alkaline hydrolysis used in the preparation of the polar given (V-hydroxy-AAF. These have not yet been studied dyes. By the use of other procedures, Kettercr and Chris- in detail. todoulides (72) have obtained evidence for a cysteinyl dye derivative in the liver protein of rats given 3'-methyl-4dimethylaminoazobenzene. Very recent studies with Dr. Lin* have shown that the nucleic add-bound derivatives formed from MAB in the oocms -c^utoff,^ =y,C0CHS ^ (OOCHj N-ACETOXY-AAF ^^=V/C0C"54 rat liver in oioo are also derived from esters of (V-hydroxy- MAB or derivatives with similar reactivity. Thus, degra dation of the RNA and DNA from the livers of rats given injections of MAB-prime ring-sH yields derivatives which + ucTHioHVLaurciNe ^ --L~\^C0CM3 3H are chromatographically identical with the compounds AH- prepared by reaction of /V-benzovloxy-MAB with guanosine or deoxyguanosine. The latter compounds have been characterized as /V-(guanosin-8-yl)-MAB and N-{deoxyguanosin-8-y IV MAB. These studies on the structures of the end products (I) ,COCHj / HgC^ ^C-^NH-CHgCOOH CHj-0 formed by reaction of dye derivative(s) with proteins and ^ + CM* C. * *tH-CHZCQOH nucleic acids in oioo cannot establish the identity of the reactive metabolite(s). However, the suggestion that js_____ n'NHj ester(s) of W-hydroxy-MAB are metabolic intermediates is supported by the similar reactivities of JV-benzoyloxy- -CH38-AAF MAB and the reactive metabolite(s) in vivo and by the data correlating the formation of the sulfuric acid ester of N-HYDROXY-AAF methionine HEPATIC PROTEIN-eoUNO'AAl* ` Lin. J.-K., Milkr. J. A.. and Miller. E. C. AMGuanosin-fLyl)- and /V-(Deoxygiianosin-8-yl>-/V-meihyi-4-mmoazobeniene: Components of Hepatic rRNA and DNA in Rats Given /V-MethyM-aminoaiobenzene, submitted for publication. Chart 6. The nonenxymatic reaction at pH 7 of the synthetic esr N-acetoxy-AAF with methionylgiydne to yield I- and 3-mclhytmctar- lo-AAF. homoserine lactone, and glycine and the liberation of 1- 3-methylmercapto-AAF from the liver proteins of rats given droxy-AAF (27. 92). T 564 CANCER RESEARCH VOL "Br,imu.iR|, ^1 398286 wp.iU: i i Clowes Memorial Lecture The synthetic esters of /V-hydroxy-AAF react readily with guamne residues in nucleosides, nucleotides, or nu cleic acids and, to a very limited extent, with adenine res idues (82. 102, 113,114). In contrast to the attack of alkyl ating agents at the N-7 position of guanine, studies with Dr. Kriek showed that the esters of /V-hydroxy-AAF re act at the C-8 position (82) (Chart 7). Thus, the reaction products of /V-acetoxy-AAF with guanosine and deoxyguanosine were characterized as jV-(guanosin-8-yl)-AAF and /V-(deoxyguanosin-8-yl)-AAF, respectively. Further, the enzymatic hydrolysis of the liver ribosomai RNA from rats given AAF or V-hydroxy-AAF to the nucleoside or nucleotide levels yields derivatives indistinguishable chromatographically from /V-(guanosin-8-yl)-AAF or its 5'phosphate derivative (79-81, 114) (Chart 7). These find ings are in agreement with the earlier work of Irving et al. (65) that no loss of the /V-acetyl group occurred in the binding of fluorene residues from /V-hydroxy-AAF to ribosomai RNA in the liver of the rat. Thus, it was of in terest that Kriek (79) noted subsequently that a large loss of the rif-acetyl group occurred in the binding of fluorene residues from AAF to the guanine bases in hepatic DNA in the rat. Recently, Irving and his associates (63) and Kriek (80, 81) have further analyzed the situation in the rat liver and find that the majority (about 70%) of the fluorene residues bound to the rat hepatic ribosomai RNA retain the /V-acetyl group and that the majority (about 70%) of the fluorene residues in the rat hepatic DNA do not retain the /V-acetyl group (Chart 7). The occurrence of the /V-(guan-8-yl)-2-acetylaminofluorene derivatives appears to be most readily explicable in terms of the in vivo reaction of an ester of /V-hydroxyAAF with guanine residues in the nucleic adds, and evi dence for this will be presented below. The reactive form which serves as the precursor of the nonacetylated deriva tives is not clear, but several possibilities can be outlined. The possible importance of reaction in oioo of A'-hydroxyAF with guanine residues in nucleic acids has been sug gested by Kriek (78) on the basis of the nonenzymatic, acid-catalyzed reaction of /V-hydroxy-AF with guanine residues and by King and Phillips (74) from their studies on the binding of /V-hydroxy-AF to tRNA with rat liver preparations in vitro. Esters of /V-hydroxy-AF, if they are formed in vivo, would be expected to be strong electrophilic reactants, but the study of this reaction has not been possible because of our inability to synthesize the model compounds. Collaborative studies in our labor atory with Dr. Irving showed that the O-giucuronide of /V-hydroxy-AAF reacts with nucleophiles in a manner sim ilar to that of the esters of /V-hydroxy-AAF, but at a very much slower rate (103). However, in addition to the differ ences in rate of reaction, another important difference was noted; thus, while the esters of /V-hydroxy-AAF yield es sentially only iV-(guanosin-8-yl)-AAF on reaction with guanosine, about one-third of the product formed in the reaction with the glucuronide is /V-(guanosin-8-yl)-AAF and about two-thirds is the deacetylated product /V-(guanosin-8-yl)-AF (103). Furthermore, recent studies by Irving and his associates (62) have shown that the glucu- N-[(DeOXY)GUANOSIN-8-YL]-AAF [(d)G-AAF] G-AAF (-70%) dG-AAFt--30%) G-AF(--30%) dG-AF(--70%) (ENZYMATIC HYDROLYSES) N-HYORCXY-AAF RAT > HEPATIC RNA- + ONA-BOUND FLUORENE DERIVATIVES Chan 7. The nonenzymauc reaction at pH 7 of the synthetic ester /V-*ceioxy-AAF with guanosine or deoxvguanosinc (82), and the (deoxy) guanosin-8-y! derivauves of AAF and AF found by Kriek (79-81) and Irving et at. (63) in the hepatic RNA and DNA of rats given labeled /V-hydroxy-AAF ronide of /V-hydroxy-AF is very reactive with guanylic acid; this compound is not known to be formed in vivo, but, in view of the large amounts of the glucuronide of /V-hydroxy-AAF formed metabolicaily, the presence of some of the deacetylated derivative would not be unex pected. Finally, the possibility that the initial product formed on reaction of the DNA in vivo is an /V-acetyl derivative, which is subsequently deacetylated, cannot be ignored. Evidence That the Sulfuric Add Ester of N-HydroxyAAF Is an Ultimate Reactive and Cardnogenic Derivative of jV-Hydroxy-AAF in the Liver. From 20 to 50% of ad ministered /V-hydroxy-AAF is excreted by the rat in the urine and bile as the O-glucuronide (66, 111, 145). How ever, this metabolite is similarly excreted in large amounts by other spedes, such as the mouse, hamster, and rabbit (67, 108, 145), which are much less susceptible to the car cinogenic activity of this hydroxamic add (68, 108). This fact suggested that the O-glucuronide is not the major metabolite responsible for the cardnogenic activity and in vivo reactivity of /V-hydroxy-AAF in the rat and prompted a search for other metabolic forms. In view of the short half-lives of the esters of /V-hy droxy-AAF in water, e.g., about 7 min for /V-acetoxy-AAF and less than 1 min for AAF-/V-sulfate at 37 (114, 138), it did not appear feasible to attempt the isolation of such esters from tissues or excreta. However, their high reac tivity did make it feasible to capture the esters by reaction with nucleophiles as they were formed in vitro. In this manner evidence was obtained by King and Phillips (74) and in our laboratory with Dr. DeBaun (27, 28) for the formation of the sulfuric add ester of /V-hydroxy-AAF by soluble liver proteins. Thus, incubation of soluble rat liver proteins with /V-hydroxy-AAF, 3'-phosphoadenosine 5'phosphosulfate, Mg+* or Mn", and methionine resulted MARCH 1970 398287 565 ST0044I75 * ^ ,STOOi i, I 76 imts A. Miller ASSAY OF ACTIVE FORMS IF AAF H VIVO N-HYDROXY -RAF SULFOTRaNSFERASE ASSAY SOLUBLE Q^<c- LIVER FRACTION : 'oti -M'APStS'-RHOSRW o-s?-o. AOCNOSINE-3'PHOSPMOeuLFATEI MS,* i- SO. 3:M b x* IN VIVO V LI VCR HOMOGENATE --------- (PROTEIN-BOUND I* AND 3-<MCTHION-S-rL 1'AAf) COLO METHIONINE -- HOMOSERINE i l** Q^K (D -CH,S-AAF (DETERMINED BY QLCI Chart 8. The m vitro assay system for /V-hydroxy-AAF sulfotranserase in the soluble fraction of rat liver and the release of I- and 3nethyimercapto-AAF from the liver protein of rats given ,V-hydroxy- AAF (27, 18). Table 3 Comparison of hepatic ,V-hvdroxy-AAF sulfotronsferase actimiv and the in cioo formation of hepatic protein-hound methionvI-AAF derivatives in animals susceptible and resistant to hepatocarcmogenesu by N-hydrxnry-AAF o-Methylmercapto-AAF from Species and sex Liver sulfotransferase in vitro* Lig/0.04 ml su- pematani/30 min) Liver protein in vivo' Lg/S g liver) Hepatocarcinogenicity of /V-hydroxyAAF* Rat (M) Rat (F) Mouse (M) Hamster (M) Rabbit (M) Guinea pig (M) 23 2 (13)* 4 0.5 (3) <0.5(3) <0.5(3) 2 0.5(3) <0.3(3) 38 9(17) 4 1 (5) <1-5 (3) <0.3 (3) <0.5 (3) <0.3 (3) +++++ + + + -- * Hepaiocarcinogemcuy dau from References 68. 108. 109. ' Assays were carried out oo liven of untreated animals. 'Animals were given i.p. injections of 5 mg /V-hydroxv-AAF in 0.5 ml dimethyl sulfoxide-com oil (1:6) emuision/100 g body weight and were killed 16 hr later. * Dau are given as average values I S.D. with the number of analyses in parentheses. in the accumulation of 1- and 3-methylmercapto-AAF, while little or none of these products was detected when 3'-phosphoadenosine 5'-phosphosulfate was omitted from the medium (27, 28) (Chart 8). 3'-Phosphoadenosine S'-phosphosulfate is recognized as a specific sulfate donor in the enzymatic sulfation of various physiological hydroxy compounds (133). In order to assess the possible importance of the sulfuric acid ester in the carcinogenic activity and in vivo reactivity of /V-hydroxy-AAF, correlative studies were carried out with Dr. DeBaun on (a) the hepatic sulfotransferase ac tivity (measured by the above assay with limiting amounts of sulfotransferase), (b) the amounts of 1- and 3-methyl mercapto-AAF which could be released from the liver protein of animals given. intraperitoneal injections of a standard dose of /V-hydroxy-AAF, and (c) the hepatocarcinogcnicity of /V-hydroxy-AAF under a variety of con ditions. Strong correlations were obtained between these 3 parameters (27). Thus, the liver of the male rat is far more susceptible to the carcinogenic activity of /V-hy droxy-AAF than are the livers of female rats or the liven of male animals of a number of other species. Similarly, the male rat liver has higher levels of sulfotransferase activity for ;V-hydroxy-AAF and higher levels of proteinbound methionyl-AAF derivatives than the livers of the other species studied (Table 3). Hepatic carcinogenesis in the male rat by AAF and its derivatives is susceptible to modification by hormonal manipulation (46, 131, 166). With male rats, thyroidectomy, hypophysectomy, or cas tration plus administration of estrogen all markedly in hibit hepatic carcinogenesis by AAF and N-hydroxyAAF. Each of these endocrine alterations also lowers the sulfotransferase activity for /V-hydroxy-AAF and reduces the amount of protein-bound methionyl-AAF derivatives in the livers of rats given /V-hydroxy-AAF (Chart 9) (27). The correspondence between the in vitro sulfotrans ferase activity and the in vivo reactivity suggests, but does not prove, that AAF-W-sulfate is a major reactive form of /V-hydroxy-AAF in the liver. This premise is also strength ened by our inability to detect appreciable amounts of a MALE RAT LIVER 40 30 < *o 6 20 o A lOH PROTEIN*BOUND o-CHjS-AAF IN VIVO 0 SULFOTRANSFERASE ACTIVITY IN VITRO s1 CONTROL HYPOPHY- THYROID- SECTOMIZED ECTOMIZED Chart 9. The effects of hypophysectomy and of thyroidectomy 1 adult male rats on the hepatic /V-hydroxy-AAF sulfotransferase actio and on the amount of hepatic protein-bound methionyl dcrivalMiJ formed in vino from /V-hydroxy-AAF (27). The endocrine organa ' removed 5 weeks before the assays were carried out. The protein-boaMj methionyl derivatives are expressed as g o-methylmercapto-AAF i leased/5 g liver 16 hr after an i.p. injection of 5 mg /V-hydroxy-AAF/H g body weight. The sulfotransferase ictivities were determined oa 1 livers of rats not given /V-hydroxy-AAF and are expressed as o-methylmercapto-AAF formed/0.04 ml liver supernatant/30 min. 566 CANCER RESEARCH VOL. Marc 398288 Clowes Memorial Lecture Tiblc 4 Stimulation by sulfate ions of the binding in moo of AAF residues to protein-bound methionine in the lioers of mate rats gwen injections of N-hydroxy-AAF The injecuon schedules were as follows. Experiment I: p-hydroxyacetamiide in I m NaO or 0.5 m Na,SO. solution was injected at 0 and 3 hr. /V-hydroxy-AAF was injected at 4 hr. Experiment 11: p-hydroxyacetanilide in 2 w NaCI or I m NatSO. solution was injected at 0 hr. and A'-hydroxy-AAF was injected at 4 hr. Experiment III: water. 1.5 m NaCI + 0.25 M MgCli solution or 0.75 m NatSO. + 0.25 m MgSO. solution were injected at 0 hr, and JV-hydroxy-AAF was in jected at 3 hr. Experiment IV: injection schedule as for Experiment 111. except that A'-hydroxy-AAF was injected at 2 hr. All injections were inirapemoneai (0.5 ml/100 g body weight). /V-Hydroxy-AAF was suspended in 1.75% gum acacia, which in Experiments 1. 111. and IV contained the same concentrations of salts as the solutions injected at 0 time. Experi ment no. p-Hydroxyacetanilide injected (to tal) (mg/100 g body weight) V-Hydroxy-AAF (mg/100 g body weight) Time rats hilled (hr after N-hydroxy-AAF in- jecuon) ug o-methylmercapto-AAF liberated/5 g liver from rats given injecuons of: Sulfate Chloride No anion I 38 II 19 III 0 IV 0 5 5 12 20 * Mean 1 S.O.; number of rats is in parentheses. 16 16 2 2 29 6 (6)' 38 * 8 (5) 78 19 (13) 98 dr 14 (4) 8 4 (6) M * 3 (6) 55 13 (7) 63 * 19 (4) 51 23 (6) 68 12 (4) phosphotransferase (27) or acetyltransferase for A'-hy droxy-AAF in rat liver. Further evidence that the sulfuric acid ester of /V-hydroxy-AAF is involved in its reactivity in rat liver in vivo was obtained in experiments in which rats were depleted of sulfate ion by administration of ^-hydroxyacetanilide [on the basis of the experiments of Biich et at. (13)]. In these animals, supplementation with sulfate ion prior to the administration of A'-hydroxy-AAF resulted in 2 to 3 times greater levels of protein-bound methionyl-AAF derivatives in the liver than if no sulfate ion was administered (29) (Table 4). When A'-hydroxyAAF-9-|4C was administered, the overall amounts of protein-bound and RNA-bound fluorene derivatives in the livers were approximately twice as great in rats ad ministered both p-hydroxyacetanilide and sulfate ion as in control rats given p-hydroxyacetanilide and chloride ions. The level of DNA-bound fluorene derivatives was approx imately one-third greater in the sulfate-treated rats than in the controls (Table 5). These treatments with p-hydroxyacetanilide and salt solutions did not alter the ,V-hydroxy-AAF sulfotransferase activity of the liver, and the levels of bound fluorenyl derivatives thus appear to reflect the availability of sulfate for the synthesis of AAF-A'-sulfate. Furthermore, when very high levels of A'-hydroxyAAF were given, injection of sulfate ion, without prior ad ministration of p-hydroxyacetanilide, caused approxi mately 50% increases in the amounts of protein-bound methionyl-AAF derivatives. Carcinogenic Activity of Esters of /V-Hydroxy-AAF and Related Hydroxamic Adds. When ultimate carcinogenic metabolites can be isolated or synthesized, it is to be ex pected that they will exhibit reactivity in the proper tests and that, under favorable conditions, these metabolites will be more cardnogenic and will be carrinogenic in a wider range of species and tissues than the parent precarrinogen. However, it is also apparent that an ultimate reactive metabolite may not show the expected cardno genic activity when administered exogenously if its reac tivity inhibits its entry into the cells. This problem has Table 5 Stimulation by sulfate ions of the binding in oioo offluorene residues to total protein, nbosomaI RNA, and DNA in lioers of mate rats p-Hydroxyacetanilide (19 mg/100 g body weight) in 2 M NaCI or 1 m Na,SO, solution was injected into adult male rats at 0 br. and /V-hydroxy-AAF-9-`*C (5 mg/100 g body weight) was injected at 4 hr. All injections were intraperitoneai (0.5 ml/100 g body weight); the (V-hydroxy-AAF was suspended in 1.75% gum acacia solution. Fluorene residues bound (pmoles/mg) of: Protein Ribosomal RNA DNA Rats pretreated with p-hydroxyaceiamlide and sulfate ions Rats pretreated with p-hydroxyacetanilide and chloride ions 1470 : 220* 325 90 280 95 780 dr 140 155 dr 70 200 70 * Mean I S.D.; 5 rats/group. The differences between the amounts of bound fluorenyl derivatives for the livers of rats given injections of sulfate or chloride ions had p values of <0.05 for DNA and ribosomaj RNA and <0.01 for protein. already been encountered in tests with the cardnogenic alkylating agents, and it appears to be exemplified again in the tests on the cardnogenidty of AAF-A'-sulfate and of several synthetic neutral and lipid-soluble esters of /V-hydroxy-AAF. Thus, while AAF-/V-sulfate is strongly implicated in hepatocarcinogenesis with A'-hydroxy-AAF, this ester has induced very few tumors on application to the skin and subcutaneous tissue of the rat.4 It seems likely that the high reactivity of this ester and possibly also its ionic nature preclude the entry of sufficient amounts of the compound into the cell before it reacts with extra cellular or cell membrane components. The situation is more favorable with the neutral and lipid-soluble synthetic esters such as Af-acetoxy-AAF and A'-benzoyloxy-AAF. These compounds, with somewhat longer half-lives, havt * E. C. Miller, and J. A. Miller, unpublished dau. march 1970 567 $ T0044 I 77 -.UUUW 398289 James A. Miller lOOr SUBCU. INJ. - IT-20 MALE RAT5/GROUP lOOr O tr 50H y* N-8x0-MAB / (285 JJMOLES) LMAB I '__ I__ I----1----1----L 36 9 MONTHS Chart 10. The inducuon of sarcomas at the site of repeated s.c. in jections of various fluorene and aminoazo dye derivatives in male rats (the total doses are given in parentheses). and Szybalski (96). The mutagenic system chosen wag that of Freese and Strack (41), in which naked transform ing DNA from wild type Bacillus subtiiis is exposed to the test compound in oitro, reisolated free of the compound, and used for the transformation of a strain of B. subtiiis which lacks gene B for tryptophan synthetase (Chart 12). Gene B is closely linked to the genes which code for the synthesis of indole from anthranilic add, and mutations in the latter genes are readily detected by the fluorescence of the anthranilic add and l-(o-carboxyphenylamino)-l-deoxyribulose vHch accumulate in the mutant cells. Hence, the frequency of transformants is a measure of the sur vival of gene B, and the frequency of fluorescent colonies is a measure of the mutagenidty of the compound to which the transforming DNA was exposed in vitro. Tht esters of Af-hydroxy-AAF inactivated and caused muta tions in the transforming DNA, and these 2 biological ef fects were proportional to each other (Chart 13) and to the extent of reaction of the esters with the DNA as deter mined from the density of the treated DNA or its content of 14C from jY-acetoxy-AAF-9-uC. (V-Benzoyloxy-MAB was likewise mutagenic in this system (96). AAF, AF, and their metabolites 2-nitrosofluorene, A'-hydroxy-AAF, jY- SUBCU. INJ. - 16 MALE RATS/GROUP 001- . COCH, proven to be stronger carcinogens than N-hydroxy-AAF at sites of application, especially the subcutaneous tissue of the rat (114) (Chart 10). iV-Hydroxy-AF and 2-nitrosofluorene were also less carcinogenic than JV-acetoxy-AAF and jV-benzoyloxy-AAF under these conditions, and sim ilar doses of AAF and AF induced no tumors at the site of injection. The O-glucuronide of N-hydroxy-AAF also has little or no carcinogenic activity under these condi tions (103). Other esters of hydroxylamines and hydroxamic acids have also shown greater carcinogenic activity at the site of subcutaneous injection than the parent amines or hydrox amic acids. Thus, jV-benzoyloxy-MAB is carcinogenic at the subcutaneous site while MAB is inactive (124) (Chart 10). Likewise, the synthetic N-acetoxy derivatives of 4-acetylaminobiphenyl, 2-acetylaminophenanthrene, and 4-aceiyiaminostilbene have each induced more tumors at the site of subcutaneous injection in rats than the corre sponding iV-hydroxy metabolites (Chan 11) (114). On the other hand, the more reactive (less stable) and ionic sul furic acid ester of jV-hydroxy-4-acetylaminobiphenyl has exhibited little or no carcinogenic activity under these conditions. Mutagenic Activity of jV-Hydroxy Esters. Since heritable alterations in the information coded in the DNA of a cell could be expressed phenotypically by a partial or complete loss of control of its multiplication, somatic mutations form a reasonable theoretical basis for the induction of tumors. Accordingly, an analysis of the mutagenic activities of various metabolic and synthetic derivatives of AAF and MAB was undertaken in collaboration with Drs. Maher 12 O * MONTHS Chart 11. The induction of sarcomas in male rats at the site of pcaied subcutaneous injections of various iV-aoetoxy and /V-hydrmJ| aromatic amides: data are also included for the sulfuric add ester < V-hydroxy-4-acetylaminobiphenyl (the total doses are given in theses). 568 CANCER RESEARCH VOL 8imoois. -'i r "(Jumpi y* 398290 Clowes Memorial Lecture TREATED DONOR (PROTOTROPH) DNA WITH ARYL AMIDE RESIDUES (AR) TRANSFORMING DNA + ESTERS OF N-HYOROXY ARYL AMIDES V AR AR AR RECIPIENT DNA (TRY') PRPP ----------|---------- ---------- K-- BA -->AWTHRANILIC -->I-(o-CARBOXYPHENYLAMINO)-I- ACIO (D DEOXYRIBULOSE-5-PHOSPHATE TRYPTOPHAN INDOLE GLYCEROL-3-FHOSBtATE \ A L- SERINE \ -------------- ---------------------- INDOLE Chan 12. Procedure for the detection of United mutations in the tryptophan operon of B. tubtilis transforming DNA by the method of Freese and Straclc (41). , , . and indicate the linked genes and the enzymes for which they code (17). The recipient cells of B. subiitis carry an inactivating mutation in gene 4B and require transforming DNA from the wild type cells to grow in the absence of tryptophan. If the piece of transforming DNA extends into regio. s , . or and, by chance, also carries a mutation in these regions, the transformed cells will grow in the absence of tryptophan only if supplemented with indole and will also accumulate fluorescent intermediates (anthramlic tad and l-(carboxyphenylamino>-l-deoxyribulose|. hydroxy-AF. the glucuronide of rV-hydroxy-AAF, and MAB yielded little, if any, inactivation or mutation of the transforming DNA. Similar studies also showed that esters of A'-hydroxy4-acetylaminobiphenyl, jV-hydroxy-4-acetylaminostilbene, and JV-hydroxy-2-acetylaminophenanthrene caused inac tivation of and mutations in transforming DNA, while the parent compounds were inactive in this test (95). In all of these cases, the chemical reactivities of the esters paral leled their inactivating and mutagenic effects on the trans forming DNA. The nature of the reactions which cause these muta tional events in transforming DNA is not clear. The majority of the mutants in these studies were spontane ously revertible at rates of 1 X lO-' to 1 X 10"', and these mutations were probably due to single base-pair changes. Since the esters attack guanine most readily, GC to AT changes would be expected. However, AT to GC mutants were the type most frequently observed in the studies of Corbett, Dove, and Heidelberger (24) on reac tion of iV-acetoxy-AAF with T4 phage. Hence, the quan titatively minor attack of N-acetoxy-AAF on adenine (114) may sometimes be the more biologically significant reac tion. On the other hand, the introduction of a bulky aryl group into the 8-position of guanine residues in DNA [as demonstrated for esters of JV-hydroxy-AAF and of N-hydroxy-MAB (82)'] might cause some local denaturation of the double helix and inhibit or block the progress of DNA polymerase along the double helix. Indeed. Troll and his associates (156, 157) have shown that DNA which has been extensively reacted with jV-acetoxy-AAF is a poor template for DNA and RNA polymerases. Interference with DNA polymerase would presumably lead most read ily to deletion mutants. Deletion mutants were obtained by Fahmy and Fahmy (36) on treatment of Drosophila with esters of /V-hydroxy-AAF and may also account for some of the nonrevertible mutants observed in our studies (95, 96). Several investigators (35, 63, 153, 164) have recently noted the persistence for many weeks of bound residues of carcinogenic aromatic amines and amides in hepatic DNA in the rat. These residues may have important ge netic consequences in carcinogenesis by these agents in the rat liver. From these studies, it is apparent that metabolically de rived esters of the carcinogenic hydroxylamines and hydroxamic acids might alter DNA in a manner leading to a heritable loss of growth controls. However, it must be emphasized that the mutagenic activities of these esters are a consequence of their electrophilic reactivities and are not necessarily related to their carcinogenic activities. Possible Epigenetic Consequences of Reactions of A-Hy droxy Esters mi Vivo. Heritable and quasi-irreversible changes presumably occur in cellular differentiation in multicellular organisms without alterations in genic infor mation. Carcinogenesis may consist of similar relatively permanent changes in gene expression. Thus the epige netic consequences of reactions of the /V-hydroxy esters of amines and amides with amino acids (e.g., methionine, cysteine, tyrosine, and tryptophan) in proteins (repres sors?) and with guanine (or other bases) in RNA's must also be considered as possible bases for carcinogenesis. The idea that such reactions could result in alterations in Chin 13. The mutagenic action of various esters of A,-hydroxyAAF on transforming DNA for B. subtilii (96). The mutagenicity index is the ratio of the mutation rate for the colonies derived from cells transformed with the reacted DNA to that of the control rate (/.., 1 X 10'*). The mutation rale for /V-acetoxy-AAF parallels the degree of inactivation (lethal hits) of the DNA and the extent of reaction of Af-acetoxy-AAF-9-'*C with the DNA. ST0044 I 79 march 1970 569 398291 James A. Miller gene expression which might be early, and possibly re versible, events in carcinogenesis was proposed by Jacob and Monod (69, 117). The theoretical basis of this sug gestion was developed in greater detail by Pitot and Heidelberger (123). At about the same time, Loeb and Gelboin (90. 91) provided experimental evidence for rapid epigenetic effects of methylchoianthrene in rat liver: Gel boin (44) later discussed these and other data in detail. More recently, Weinstein (165) has studied the biochem ical effects of reaction of tRNA's with N-acetoxy-AAF. From these and other studies, he has proposed that tu mors may result from the effects of potentially reversible aberrations in differentiation which arise through carcin ogen-induced modifications of tRNA's. ULTIMATE CHEMICAL CARCINOGENS AS STRONG ELECTROPHILIC REACTANTS The foregoing studies on the reactivity of the esters of /V-hydroxy amines and amides and a consideration of the literature on the known or postulated reactive forms of other chemical carcinogens suggests that most, and per haps all. of the chemical carcinogens either are strong electrophilic reactants as administered or are converted in vivo into potent electrophilic reactants. It is presumed that these electrophilic reactants then initiate the carcin ogenic process through certain of their reactions with nu cleophiles in crucial tissue components such as the nucleic acids and proteins. Knowledge of the structures of the reactive forms of carcinogens is still in a preliminary stage of investigation in many cases, but the data summarized below lend strong support to this generalization. The data for a variety of aliphatic carcinogens are sum marized in Chart 14. The alkylating agents are recognized as electrophilic reactants per se, and numerous studies have documented their nonenzymatic reactivity under physiological conditions with nucleophilic sites in nucleic acids and proteins; similar reactions also occur in vivo (8. 11. 12. 21, 22. 83, 85. 127, 129, 134, 141, 167). In addi tion. chemical carcinogens of a variety of types are con verted in vivo into alkylating agents through enzymatic or nonenzymatic means. These potential alkylating agents include the carcinogenic nitrosamines and dialkylaryltriazenes which are activated by enzymatic dealkylation and the nitrosamides which are activated nonenzymatically through reaction with sulfhydryl groups (31, 32, 86, 87, 94, 126, 128, 139). As Laqueur has shown (84), the naturally occurring carcinogen cycasin is hydrolyzed by bacterial 0-glucosidase in the intestinal tract to the aglycone methylazoxymethanol. This compound methylates nucleic acids in vivo (119. 139) and in vitro (98). Ethylation of RN A and proteins in the rat liver occurs as a consequence of the ad ministration of ethionine (37, 39, 122, 147, 148). The dem onstration that the alkylating agent 5-adenosylethionine is formed in the liver of the rat after administration of ethi onine led to the assumption that this compound was the intermediate responsible for the ethylation of proteins and nucleic acids. However, the more recent studies of Ort- CARCINOGEN (PRECARCINOGEN) combounos yielding ALKYLATING AOCWTa . QIALKYLBITBOSAMINES CM3, ^ N-NO POSSIBLE CARCINOGENIC ELECTROPHILIC REACTANT (ULTIMATE CARCINOGEN) ALKYLATING AGENT* wcACTiooui ! ~ i HgC--e=o CM, CjMj0~C=0 ClfiASlfi. - HJB * CHj-M^M-CMjO-a-CtUCOSVL--CM^M=N-CNzOH---- ^ CHj i MZ' 0 ctwomiwe fVHf CjMj-s-chjCMjCm-cookI AOCNOSYL CjM, BYBMQLIZIOINC ALKALOIDS b'-o cw-o-co-ft 03 _ + :tf-OCH^rO-CO-B CNZ. CE>` UflCTtlAh. C^M-jO-CO, -WM*j 7--* W-60-""! tt'WctM50-co-MM0H-L5re5TEiwT CAMION TETBACHLOBIOC CjHjjO-CO-tB* ect, 1 > CClj-, tCCt,) Chart 14. Ultimate carcinogenic electrophilic reactants that may he formed in oioo from various aliphatic carcinogens. werth and Novelli (122) have led to the suggestion that some other alkylating agent is also formed from ethionine in rat liver. While the highly carcinogenic pyrrolizidine alkaloids have weak alkylating activity per se. Mattocks (101) and Culvenor et al. (26) have shown that these com pounds are converted in vivo to much stronger alkylating agents through enzymatic dehydrogenation to pyrrole de rivatives. The versatile carcinogen urethan or its JV-hydroxy metabolite apparently yield several kinds of reactive electrophiles including free radicals (70, 116, 120) which may react with cysteine (Chart 14) and with cytosine in RNA in vivo (10). Evidence for the formation of alkylating derivatives of carbon tetrachloride comes from the demon stration of protein- and, possibly, of nucleic acid-bound forms in rat liver in vivo by Reynolds (132); possible inter mediates are a free radical or a carbonium ion (15, 40, 144). The known or presumed electrophilic forms of various aromatic carcinogens are similarly shown in Chart 15. The carcinogen JV-nitroso-N-phenylurea decomposes in water within a few minutes to phenyl diazonium ion (126), and it seems certain this product must also be formed in vivo. The potent carcinogen 4-nitroquinoline-l-oxide is reduced enzymatically in rat liver and subcutaneous tissue to 4-hydroxyaminoquinoline-1-oxide (99), which is a more potent carcinogen than the parent compound (33, 142, 143). By analogy with studies on TV-hydroxy-AAF, esterification of this N-hydroxyamine in vivo might be expected. While CO i~ 570 CANCER RESEARCH VOL # 398292 Clowes Memorial Lecture no direct data are available, we have shown with Drs. Enomoio and Sato that the synthetic diacetyi derivative 0f 4-hydroxyaminoquinoline-l-oxide reacts with various nucleophiles at neutrality (34). Furthermore, in our stud ies, reaction of the diacetyl derivative with DNA in vitro yielded a fluorescent derivative with properties very sim ilar to those reported by Tada et al. (154) and Matsushima et ai. (100) for the DNA isolated from ascites cell tumors from rats treated with the N-hydroxy derivative. The car cinogenic iV-hydroxy derivatives of various purines (151) may also undergo esterification in vivo. Thus, as shown re cently by Stohrer and Brown (150), 8-chloro- and 8-methylmercaptoxanthine are excreted in the urine of rats treated with 3-hydroxyanthine; these same products are formed by reaction of 3-acetoxyxanthine with chloride ion or with methionine in vitro (172). Even aflatoxin B,, which is a very strong hepatocarcinogen in the rat (171), appears to require activation for carcinogenic activity. Thus, in view of the high susceptibility of the livers of hypophysectomized rats to carcinogenesis by dimethylnitrosamine (86), the refractoriness of such rats to aflatoxin Bi-induced liver tumors (46, 47) suggests that they do not metabolize the aflatoxin efficiently to ultimate carcinogenic forms. The reactions involved in the activation and binding of the carcinogenic polycyclic aromatic hydrocarbons to pro tein (56) and nucleic acids (12) in vivo have not been elu- CARCINOGEN (PRECARCINOGEN) POSSIBLE CARCINOGENIC ELECTROPHILIC REACTANT (ULTIMATE CARCINOGEN) COMPOUNDS nCUOINS artlatins , aryuamcatins, AMO RELATED ASCATS AROMATIC AmWES A-i)PHVATlYtt I. "-"gTOXTlATIC*, t.UTl*iriCATION 0*0 M-A~aCHjJ"-T*,AaCH^ NO Q-^-nm, 0 1M * _ HyON ^vb-(SO^T) of-^co-^cV 6o ^so,?' POLTCTCUC ASOMATIC HTDRQCARSOHS T H H HA ll RADICAL CATION CAWIHWtWC JtCTALl fc, cT.co",mT.nT cidated. Boyland (9) and his associates have strongly im plicated electrophilic epoxide derivatives as metabolic intermediates. Grover and Sims (51) and Gelboin (45) have reported the formation of reactive metabolites of the polycyclic hydrocarbons on incubation with microsomal oxidases of rat liver; the chemical nature of these reactive metabolites (epoxides?) that bind covalently to DNA, RNA, and protein in vitro is not known. Another proposal is that of Fried and Schumm (42) and of Wilk and his as sociates (168, 169), who have implicated the formation of a radical cation by a single electron oxidant on the basis of model experiments in vitro. They have postulated that the radical cation reacts with a cellular nucleophile and then undergoes a final oxidation and loss of a proton to form a stable aromatic bound form. Recent data by Ts'o and his group (159) have shown that a similar oxidation of benz(a)pyrene in the presence of DNA leads to covalent linkage of the hydrocarbon to the DNA. Chart 15 also shows ionic electrophilic forms of the car cinogenic metals. Little is known about the mechanisms by which these metals induce tumors. However, some of these metal ions are known to react with guanine (140) or to form relatively insoluble phosphates. Thus, at least 2 means of reaction with cellular nucleic acids are evident. It is evident that most of the bond-breaking reactions in living cells are helerolytic and generate nucleophiles and electrophiles. These reactions are almost always under tight enzymatic control, and the molecular fragments so generated are generally combined with other electrophiles and nucleophiles at the enzyme surfaces. In contrast, the foreign carcinogenic electrophiles are strong electrophiles which do not appear to require enzymes to facilitate their reactions with cellular nucleophiles. They can readily dis place weak electrophiles such as H+ from cellular com ponents and probably enter into reactions rather indis criminately with strong nucleophilic groups in cellular components. Some of these reactions are probably of no great consequence to cells, some must impair the functions of molecules that cells can replace, and still other reactions apparently initiate chains of molecular events which lead to losses of growth controls in cells and their progeny. The characterization of ultimate chemical carcinogens as strong electrophilic reactants brings considerable order to the confusing variety of chemical carcinogens which is now evident. From this vantage point, it is probable that there is both theoretical and practical value in research on attempts to (a) inhibit or prevent the generation in vivo of these strong electrophiles, (b) render these strong elec trophiles harmless in reactions in vivo with low-molecularweight, nontoxic nucleophiles, and (c) predict the carcino genicity of foreign molecules (e.g., drugs, food additives, food-water-air contaminants, etc.) from measures of their conversion in vivo to reactive electrophiles. Chart 15. Ultimate carcinogenic electrophilic reactant! that may he derived in vido from various aromatic carcinogens, and the possible reaction of a radical cation derived from a polycyclic aromatic hydro carbon with a tissue nucleophile. Electrophilic cations for various car cinogenic metals are listed. PERSPECTIVES From the foregoing discussions, it is apparent that stud ies in chemical carcinogenesis have proceeded to the point 571 398293 18 1*i *10 S01 a I i James A. Miller that information is becoming available on the nature of the reactive electrophilic forms of chemical carcinogens in oioo. Likewise, information is accumulating on the na ture of the nucleophilic targets of these ultimate carcino gens in oivo. However, in no case of chemical carcinogen esis do we know unequivocally the nature of the molecular target(s) critical to the induction of that carcinogenic process. Unfortunately, entirely analogous deficiencies exist in our knowledge of the molecular mechanisms of action of carcinogenic viruses and radiations and in our characterization of the molecular phenotypes of neo plasms. Lack of knowledge of the chemical nature and genetic significance of the critical molecular target(s) in all forms of carcinogenesis has severely limited progress, and only general hypotheses have been proposed for the mechanism(s) of action of carcinogens. At the minimum, carcinogenesis evidently consists of a heritable and at least quasipermanent loss of control of cell multiplication. At present, there are at least 2 general basic hypotheses that might account relatively directly for such a change in cellular behavior (Chart 16). These hy potheses are (a) interactions of carcinogens with DNA re sulting in alterations in the information contained in this macromoiecule and (6) alterations in specific proteins or RNA's which result in relatively stable and heritable changes in genome expression. In addition, at least 2 in direct mechanisms have been proposed which are likely to operate through one of the previous mechanisms, i.e., the activation of a latent carcinogenic virus genome and the selection of preneoplastic cells by conditions that favor the multiplication of these cells. Conceivably, combina tions of these direct and indirect mechanisms may occur. Unfortunately, none of these or any other hypotheses have yet received unequivocal experimental support in any in stance with any carcinogenic agent. Fortunately, however, the rapid increases in our knowledge of cellular and mo lecular biology in the last decade give real promise that PRECARCINOGENS 'i' METABOLISM carcinogenic electrophilic reactants (ultimate carcinogens) V* R_A ------------------^ R +A T^ nucleophiles in critical -f CELLULAR TARGETS: BASES IN NUCLEIC ACIDS AMINO ACIDS IN PROTEINS OTHER CELLULAR COMPONENTS altered, N> UCLEIC ACIDS OR PROTEINS OR BOTH CEHET1C EFFECTS ^ _ epigenetic effects DIRECT: INDIRECT; MUTATIONS ACTIVATION OF^ VIRUS - / NEOPLASIA CHANGE IN GENOME EXPRESSION SSELECTION OF LATENT TUMOR CELLS Chtri 16. 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