Showing papers in "Cancer Research in 1967"

The Detection of Disease Clustering and a Generalized Regression Approach

Abstract: The problem of identifying subtle time-space clustering of disease, as may be occurring in leukemia, is described and reviewed. Published approaches, generally associated with studies of leukemia, not dependent on knowledge of the underlying population for their validity, are directed towards identifying clustering by establishing a relationship between the temporal and the spatial separations for the n ( n - 1)/2 possible pairs which can be formed from the n observed cases of disease. Here it is proposed that statistical power can be improved by applying a reciprocal transform to these separations. While a permutational approach can give valid probability levels for any observed association, for reasons of practicability, it is suggested that the observed association be tested relative to its permutational variance. Formulas and computational procedures for doing so are given. While the distance measures between points represent symmetric relationships subject to mathematical and geometric regularities, the variance formula developed is appropriate for arbitrary relationships. Simplified procedures are given for the case of symmetric and skew-symmetric relationships. The general procedure is indicated as being potentially useful in other situations as, for example, the study of interpersonal relationships. Viewing the procedure as a regression approach, the possibility for extending it to nonlinear and multivariate situations is suggested. Other aspects of the problem and of the procedure developed are discussed. Similarly, pure temporal clustering can be identified by a study of incidence rates in periods of widespread epidemics. In point of fact, many epidemics of communicable diseases are somewhat local in nature and so these do actually constitute temporal-spatial clusters. For leukemia and similar diseases in which cases seem to arise substantially at random rather than as clear-cut epidemics, it is necessary to devise sensitive and efficient procedures for detecting any nonrandom component of disease occurrence. Various ingenious procedures which statisticians have developed for the detection of disease clustering are reviewed here. These procedures can be generalized so as to increase their statistical validity and efficiency. The technic to be given below for imparting statistical validity to the procedures already in vogue can be viewed as a generalized form of regression with possible useful application to problems arising in quite different contexts.

Hydroxyurea: Effects on Chinese Hamster Cells Grown in Culture

Abstract: Summary Hydroxyurea has both inhibitory and cytotoxic action on Chinese hamster cells grown in vitro. During short exposures, the lethal action is selective for cells in DNA synthesis (S cells) only. The inhibitory effect prevents incorporation of tritiated thymidine into DNA while RNA and protein synthesis continue essentially without modification, at least until near the end of the cycle. S cells, destined to die, fail to divide after exposure to hydroxyurea, but resynthesize DNA after the drug is removed, enlarge to 3–4 times the normal volume during the next 20–30 hr, and then lyse. Cells at stages of the cell cycle, other than S, progress normally until the time DNA synthesis should have begun. A condition of unbalanced growth then ensues, which cells tolerate quite well for some hours, and from which they recover when relieved. They cannot remain viable in this condition indefinitely, however, and die after being inhibited for about 8–10 hr (i.e., about the time the next division should have occurred). The lethal and inhibitory actions of the drug make it a useful synchronizing agent in asynchronous populations, even though the length of the S period may be slightly reduced thereafter. Furthermore, although inhibited cells are more sensitive to X-rays in the presence of the drug, they recover normal X-ray responses within a short time after the drug is removed.

Cellular Analysis of Liver Carcinogenesis: the Induction of Large Hyperplastic Nodules in the Liver with 2-Fluorenylacetamide or Ethionine and Some Aspects of Their Morphology and Glycogen Metabolism

Abstract: Summary A dietary regimen is described whereby large hyperplastic nodules can be obtained in over half the rats fed either of the two hepatocarcinogens, ethionine or 2-fluorenylacetamide. The nodules are composed predominantly of hepatocytes which show morphologic and biochemical differences from the surrounding nonhyperplastic liver. The glycogen or its metabolic control are different in the nodule in that a significant amount of glycogen is present even after a 48-hour period of fasting and little breakdown is induced by glucagon administration. The nodules show a consistent and progressive decrease in glucose-6-phosphatase activity as well as in glycogen phosphorylase activity. Evidence implicating the hyperplastic nodule as a step in the carcinogenic process is presented and discussed.

Zinc and magnesium in human prostate gland: normal, hyperplastic, and neoplastic.

Abstract: Summary Using histochemical and biochemical (atomic absorption spectrophotometer) methods, a study of normal prostate glands disclosed a fundamental difference in distribution of zinc in the various zones and a significant difference in concentration in pathologic conditions. Magnesium in the normal prostate showed a uniform distribution and concentration, and both elements (Zn++ and Mg++), especially zinc, showed multifold increases in concentration in the hyperplastic glands. In carcinoma, zinc was present in cell nuclei corresponding to nucleolar position, and chemically the content was low. Magnesium was not demonstrable histochemically in carcinoma, and the chemical assay showed lower concentration than in hyperplastic tissue. The most significant findings were the chemical and histochemical comparable multifold increases of zinc and magnesium in the hyperplastic gland. In addition, there was a marked decrease of zinc histochemically and chemically in carcinoma, while the chemical assays for magnesium disclosed a minimal decrease; this element was not demonstrable histochemically in cancer tissues.

Hydroxyurea. I. Acute cell death in proliferating tissues in rats.

Abstract: Summary Hydroxyurea induces karyorrhexis in intestinal crypts, bone marrow, and germinal centers of all lymphoid tissues within a few hr after injection. Acute, selective cytotoxic effects are limited to those tissues with high rates of cellular proliferation. The pathologic process is of short duration, and repair of tissue damage is rapid. Other hydroxamic acid derivatives induce similar lesions: N-hydroxyurethan, acetohydroxamic acid, N,O-diacetyl-N-methylhydroxylamine, and 1-methyl- and 1-ethyl-1-hydroxyurea. The lesions are not seen in rats given hydroxylamine or N-methylhydroxylamine. The cytotoxic effects are related in time to the physiologic disposition of hydroxyurea. The agent is rapidly equilibrated throughout body water. Its concentration in plasma decays exponentially with half-lives of 65 min at levels above 150 μg/ml and of 35 min below 100 μg/ml. N-Hydroxyurethan and acetohydroxamic acid are disposed of in vivo at comparable rates. All 3 substances are excreted unchanged in urine and are also extensively metabolized. The close temporal relation between the build-up and repair of cytotoxic changes and the physiologic disposition suggests that the proximal biochemical defect produced by the hydroxamates is reversible. Hydroxyurea induces an immediate inhibition of thymidine incorporation into the DNA of thymus and small intestine in rats. The inhibition is reversible; its duration varies directly with the dose of hydroxyurea. From information about tissue concentrations it can be concluded that the sensitivity of thymidine incorporation in intestine and thymus in vivo is close to that of HeLa cells in culture. The cytotoxic potency of hydroxyurea, N-hydroxyurethan, and acetohydroxamic acid in intact rats is in proportion to their relative activity in inhibiting thymidine incorporation in HeLa cells. It is concluded that the lethal effects of the hydroxamates are restricted to cells committed to DNA synthesis. Other cells in proliferating tissues escape damage during the limited time of circulation of inhibitory concentrations; this accounts for the prompt repair of tissue defects.

Inhibition of DNA Synthesis in HeLa Cells by Hydroxyurea

Abstract: Summary Hydroxyurea is a highly specific, rapidly acting inhibitor of DNA synthesis in HeLa S3 cells. It is without detectable effect on cells that are not synthesizing DNA. Incubation in inhibitory concentrations for as long as 19 hr does not kill cells in any phase of the division cycle. Rapid and complete reversal is accomplished simply by removing the drug from the culture medium.

Kinetics of Cell Proliferation of an Experimental Tumor

Abstract: Summary The cellular proliferation kinetics of an experimental fibrosarcoma in C3H mice have been studied in vitro and in vivo at various stages in tumor growth. The duration of the cell cycle measured in vitro is the same as that measured in vivo and does not change when the increase in cell number, at first exponential, slows progressively. The slowing down of the growth rate and the plateau in vitro are explained mainly by a reduction in the proportion of cells engaged in the cell cycle and by increasing cell death. In vivo, the growth rate is at first rapid and then slows progressively. The duration of the cell cycle is similar in all phases of tumor growth. A diminution both of the number of labeled cells after multiple injections of tritiated thymidine and of the growth fraction is seen as the growth rate slows. It is probable that in this case also increasing cell death contributes to the slowing of tumor growth. Autoradiographs in large tumors after multiple injections show considerable heterogeneity in labeling from one region of the tumor to another.

Hydroxyurea-induced inhibition of deoxyribonucleotide synthesis: studies in intact cells.

Abstract: Summary Hydroxyurea-induced inhibition of thymidine incorporation by monolayers of HeLa cells was partially prevented and reversed by addition of deoxyadenosine, deoxyguanosine, and deoxycytidine to the culture medium. All 3 deoxyribonucleosides were required for optimal effect. Hydroxyurea inhibited incorporation of thymidine into DNA of embryos of the sand-dollar species Echinarachnius parma . The onset of inhibition was delayed for 3–4 synthesis ( S ) periods when the drug was added at fertilization, but was evident with the next replication cycle following drug addition between the 5th and 6th S period. Hydroxyurea did not alter rates of leucine incorporation into protein regardless of the time of drug exposure. Echinoderm ova possess significant quantities of preformed deoxyribonucleotides; previous studies with 5-fluorodeoxyuridine have suggested that these stores can supply the embryo with the thymidylate required for DNA replication over 6 S periods following fertilization. These observations, i.e., partial reversal of drug-induced inhibition by exogenous deoxyribonucleosides in HeLa cells, and the insensitivity of sand-dollar embryos to hydroxyurea during initial S periods, are compatible with the postulate that this compound inhibits reduction of ribonucleotides to deoxyribonucleotides.

Chromosomes of “Minimal Deviation” Hepatomas and Some Other Transplantable Rat Tumors

Abstract: Summary Chromosome studies were done on 35 transplantable rat hepatoma lines, including a number of “minimal deviation” tumors. Cell suspensions were obtained directly from the solid neoplasms by trypsinization, and tumor metaphases were distinguished from contaminating host metaphases by sex chromosome differences. Six tumor lines had a normal chromosome number—42—but only 9618A had a completely normal karyotype. Minimal abnormalities, which could represent normal karyotype variation, were observed in 4 others (7794A, 7800, 9098, 9121), and 9108 had definite changes involving several small chromosomes. This tumor also showed 50% transition to 43 chromosomes in a later transplant generation. The six diploid tumors had an intermediate growth rate and variable enzyme alterations. The 29 aneuploid tumors all had different karyotypes with no obvious correlation between specific chromosome alterations and specific enzyme changes. Some of these tumors were less deviated metabolically than the diploid neoplasms (e.g., 7793, 45 chromosomes) and some were slower growing (e.g., 7787, 44 chromosomes). Chromosome studies will permit identification of tumors which are “minimally deviated” from a cytogenetic standpoint, but a variety of metabolic alterations may still be present.

Infection of an established mouse bone marrow cell line (JLS-V9) with Rauscher and Moloney murine leukemia viruses.

Abstract: Summary A cell line (JLS-V9) established from the bone marrow of normal weanling BALB/c mice has been shown to be susceptible to infection with either the Rauscher or Moloney murine leukemia viruses. The Rauscher- and Moloney-virus-infected cultures were designated JLS-V10 and JLS-V11, respectively. Both infected cultures were carried for 7 months without loss of infectivity. However, more recent bioassays of Rauscher virus from the JLS-V10 line have indicated a reduction of infectivity for BALB/c mice. Virus multiplication was evaluated both by electron microscopy and bioassays of cell free tissue culture fluids. The viruses were identified by serum neutralization and response of infected mice to the respective viruses, as well as electron microscopy. Cytopathogenic effects of the viruses have not been observed in either of the infected cultures.

Glucose consumption by transplanted tumors in vivo.

Abstract: Summary The relationship between availability and utilization of glucose and production of lactate was studied in vivo in Walker carcinoma 256, Hepatoma 5123, and Fibrosarcoma 4956 transplanted in rats. The tumors utilized 0.96, 0.55, and 0.45 gm of glucose/hr/100 gm wet tumor weight, respectively, corresponding to 28%, 23%, and 32% of the amount supplied. About 35% of the glucose consumed was eliminated as lactate regardless of the tumor type. In normoglycemia, the amount of glucose consumed and glycolyzed was directly related to the glucose available which decreased per unit weight as the tumor size increased. During hyperglycemia, produced either by dextrose injections or diabetes, the tumors increased glucose utilization. The increase was maximum during the first few hours; then a “saturation” level was reached. When hyperglycemia was prolonged for several days, carcinomas and hepatomas consistently consumed about ½ more glucose; however, fibrosarcomas reduced their consumption by about 30%. Insulin did not improve glucose consumption of tumors either in normal or diabetic rats. Actually, the overall utilization was reduced because the plasma level of glucose in the host was lowered by insulin. Hypoglycemia or injections of 2-deoxy-d-glucose reduced glucose consumption, and in vivo the tumors were unable to compensate for this decrease by removing more glucose from the blood. The vascular walls of neoplastic vessels maintained a sharp concentration difference between glucose in plasma and in the interstitial fluid, with the fluid surrounding the neoplastic cells containing only a few mg percent of free glucose and practically all of the glucose passing through the vascular walls being rapidly consumed. During hyperglycemia the concentration difference disappeared and the vascular and interstitial compartments had about equal concentrations of glucose. Normo- or hypoglycemia restored the concentration gradient for glucose between the vascular and the interstitial compartment. The time needed to complete the restoration was longer after prolonged hyperglycemia. The regulation of the glucose transfer by the vascular wall of neoplastic vessels is postulated.

The antitumor activity of Cu(II)KTS, the copper (II) chelate of 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazone).

Abstract: Summary The antitumor activity of the copper(II) chelate of 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazone), Cu(II)KTS, against the Walker 256 carcinosarcoma in rats is shown, and the importance of considering this chelate as the active intermediate in the mechanism of action of the free ligand, KTS, is indicated. The potentiation of the antitumor activity and toxicity of Cu(II)KTS by KTS and Vu(II) was demonstrated. Results of experiments with combinations of these agents are consistent with the hypothesis that the dynamics of the chemical equilibria governing the interaction of KTS and Cu(II) to form Cu(II)KTS also govern their biologic activity. The protonated chelate, Cu(II)KTS·2HCl, appears to be less active as an antitumor agent than is the uncharged chelate but also somewhat more toxic for rats.

Binding of tritium-labeled polycyclic hydrocarbons to dna of mouse skin.

Abstract: Summary Evidence was obtained to confirm previous observations of the firm binding of certain carcinogenic polycyclic hydrocarbons to the DNA, RNA, and protein isolated from mouse skin after the in vivo application of these compounds. The possibility of the influence of several artifactual causes of hydrocarbon binding to DNA was carefully investigated, and it was concluded that the binding that is measured represents the true extent of metabolic reaction between the hydrocarbon and the DNA of mouse skin. The correlation between the extent of binding to DNA and carcinogenicity was studied, and two instances were observed in which binding of a hydrocarbon to DNA was not associated with a carcinogenic event. The possible relationships between these findings and carcinogenicity are discussed.

The Interaction of 7,12-Dimethylbenz(a)anthracene with Cells Sensitive and Resistant to Toxicity Induced by This Carcinogen

Abstract: The multiplication of monolayer cultures of transformed rodent cells and normal or transformed human cells was not inhibited by concentrations of 7,12-dimethylbenz(a)anthracene (DMBA) 200–500 times greater than those that inhibited the multiplication of normal embryonic rodent cells. Fluorescence microscopy showed that DMBA localized to about the same degree within the cytoplasm of cells that were sensitive or resistant to the toxic effect of the carcinogen. Autoradiography with DMBA-3H and an aqueous cell fixative (formalin) revealed that the carcinogen was distributed throughout the cytoplasm and nucleus and that the concentrations were similar in sensitive and resistant cells. However, when the cells were treated with a fixative in which DMBA is soluble (Carnoy's), most (90%) of the labeled compound was eliminated from the resistant cells whereas a considerable amount appeared to be bound within both the cytoplasm and the nucleus of sensitive cells. The difference in the binding of DMBA by the 2 types of cells was confirmed by isolating and assaying for radioactivity individual cellular constituents after cells had been exposed to DMBA-3H. Sensitive cells bound 10–50 times more DMBA to their nucleic acids and protein than did resistant cells. With mouse embryo cells, the amount of DMBA bound to DNA and protein was proportional to the growth-inhibitory effect of the carcinogen within the dose range 0.01–0.1 µg/ml.

The Effect of Dietary Mineral Supplements of the Rat on the Antitumor Activity of 3-Ethoxy-2-oxobutyraldehyde Bis(thiosemicarbazone)

Abstract: Summary The antitumor activity and toxicity of 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazone) (KTS) in rats bearing Walker 256 nitrogen-mustard-resistant carcinosarcoma (W-256-NMR) has been shown to be directly dependent on the dietary intake of cupric ion. The critical effect of environmental and dietary trace metals on KTS activity is further indicated by the fact that zinc and cobaltous ions may influence both toxicity and antitumor action of the drug when copper is present. Copper is critical for KTS activity whether the drug is given orally or intraperitoneally. Copper was shown to be without effect on W-256-NMR in the absence of drug, while zinc was shown to be necessary for W-256-NMR growth as well as rat growth.

The production of carcinoma of the urinary bladder in rats by feeding N-[3-(5-nitro-2-furyl)-2-thiazolyl]formamide.

Abstract: Thirty female Sprague-Dawley rats were fed 0.188% of N -[4-(5-nitro-2-furyl)-2-thiazolyl]formamide for 46 weeks. Twenty-nine of the rats lived 34 weeks or more and all of these animals developed gross bladder carcinomas. Three rats developed carcinomas of the renal pelvis which invaded the kidney. There were 15 animals with 1 or more gross mammary tumors, all of which were classified as benign adenoma, fibroadenoma, or adenofibroma. This is the first nitrofuran derivative which has been found to be a bladder carcinogen, and the compound appears to be one of the most effective bladder carcinogens for this species.

Control of Lipid Metabolism in Hepatomas: Insensitivity of Rate of Fatty Acid and Cholesterol Synthesis by Mouse Hepatoma BW7756 to Fasting and to Feedback Control

Abstract: We have measured the conversion of the 14C of acetate-1-14C to CO2, fatty acids, and cholesterol by slices of liver and the transplantable hepatoma BW7756, from C57L/J mice on a variety of diets: chow, fasting, refed, fat-free, high-fat, and high-cholesterol. Whereas synthesis by liver differed greatly between animals on different diets (even as much as 17-fold for fatty acid synthesis and 19-fold for cholesterol synthesis, but more generally 4- to 5-fold), synthesis by the tumor was unaffected. Although the actual mechanisms of normal dietary control of hepatic lipogenesis are not clearly known, we suggest that the insensitivity of the hepatoma to the nutritional state of the animal may be due to inability of the particular activator(s) or inhibitor(s) to gain entry into the tumor cell.

Excretion and tissue distribution of radioactivity from aflatoxin B1-14-C in rats.

Abstract: Summary Excretion patterns of 14C derived from intraperitoneal doses of ring-labeled and methoxy-labeled aflatoxin B1 were investigated in male rats. It was found that 70–80% of the administered radioactivity from both compounds was excreted during the 24 hours following administration. A major excretory route of the ring-labeled material was through biliary excretion into feces, which accounted for nearly 60% of the administered 14C. A further 20% was excreted in urine, and only negligible amounts in CO2. In contrast, approximately 25% of administered radioactivity from the methoxy-labeled compound appeared in CO2 with a concomitant decrease in feces. These results indicate that O-demethylation represents a significant metabolic pathway for aflatoxin B1 in the rat. Radioactivity derived from ring-labeled compound was present at maximum levels in liver and kidneys 0.5 hour after administration, with small amounts present in other organs. The concentration of radioactivity was five to fifteen times greater in liver than in other tissues, and at the end of 24 hours, the liver contained an amount equal to the content of the remainder of the carcass. This finding is associated with the relative tissue specificity of aflatoxin B1 as a hepatotoxin.

Effects of Dietary Fat on Mammary Carcinogenesis by 7,12-Dimethylbenz(α)anthracene in Rats

Abstract: Summary A semisynthetic high-corn oil diet enhanced the development of mammary cancer induced by 7,12-dimethylbenz(α)anthracene (DMBA) in intact female Sprague-Dawley rats. This was in comparison to two other groups of similarly treated rats fed a high-coconut oil and a low-fat semisynthetic diet, respectively. The average daily caloric intake was similar, and the average growth rate, based on body weight, was comparable in all three groups. Fatty acid analyses demonstrated that, at the time of DMBA administration, the composition of mammary fat was different in the three different groups, reflecting their dietary fat intake. The data from this study suggest that dietary effects upon DMBA mammary carcinogenesis were related to the nature as well as the amount of fat used.

Association of Embryonic Antigens with Experimentally Induced Hepatic Lesions in the Rat

Abstract: Summary Short-term hepatic injury was produced in rats by single i.p. injections of N -dimethylnitrosoamine, carbon tetrachloride, and cadmium sulfate, or by partial hepatectomy. In all cases, the animals responded with varying serum titers of embryonic α 2 -glycoprotein which could be detected as early as 24 hours after the treatment. Antigen LA, another embryonic constituent, was never detected under the above conditions. Long-term hepatic injury was produced by oral ingestion of carcinogenic aminoazo dyes, N -dimethylnitrosoamine and aflatoxin B 1 , and by noncarcinogenic analogs of aminoazo dyes. The lesions induced were those classically obtained following such treatments: hepatocarcinoma, bile-duct carcinoma, cirrhosis, hepatitis, etc. The embryonic α 2 -glycoprotein was associated with all these lesions, whereas Antigen LA was specifically associated with rats bearing hepatocarcinomas. Neither Antigen LA nor the α 2 -glycoprotein were found in rats bearing tumors of other organs than the liver. Lipoprotein-esterase was increased in the majority of sera of rats with these various hepatic lesions.

Inhibition of in Vitro Lymphocyte Transformation during Chemotherapy in Man

Abstract: Summary The in vitro transformation responses of lymphocytes to stimulation with phytohemagglutinin (PHA) and smallpox vaccine (vaccinia) were studied in cells from 20 patients with ocular and malignant diseases receiving chemotherapy. The transformation of lymphocytes to lymphoblast-like cells was reduced from 71% in the pretreatment PHA-stimulated cultures to 1.5% during therapy. The response to vaccinia was reduced from 12% before therapy to 0% during therapy. The mitotic indices fell from 1.5% (PHA) and 1.2% (vaccinia) to 0% for each during therapy. Intensive combination therapy with parenteral 6-mercaptopurine and methotrexate, with or without prednisolone completely abolished transformation after 3 days of treatment. Substantial recovery occurred within 3 days after the end of therapy. Nontoxic therapy with methotrexate or 6-mercaptopurine which did not induce leukopenia took 2–5 weeks to cause maximum suppression. The abnormality seemed due to intrinsic damage to the lymphocytes and not to persistent antimetabolite in the plasma. In vitro lymphocyte transformation is an easy and reproducible way of evaluating the immune competence of an individual9s circulating lymphocytes.

Action of Hydroxyurea on the Nucleic Acid Metabolism and Viability of HeLa Cells

Abstract: Summary Exposure of HeLa S-3 cells to 10 -3 m hydroxyurea for a period of 16 hr produced a loss of cell viability accompanied by an inhibition of DNA synthesis. Continued synthesis of RNA and protein suggests an unbalanced growth similar to that previously observed with 5-fluorodeoxyuridine, excess thymidine, and 1-β-d-arabinofuranosylcytosine. A threefold increase in thymidine kinase activity was observed as a result of unbalanced growth following the exposure of cells to hydroxyurea. When cells were exposed to 10 -2 m hydroxyurea for short exposure times, however, appreciable loss of cell viability occurred only for those cells in the DNA synthetic phase.

Effect of vitamin A on 7,12-Dimethylbenz(alpha)anthracene-induced papillomas in rhino mouse skin.

Abstract: Summary Rhino mice fed a diet containing 100 IU of vitamin A per gm of feed produced fewer papillomas than did similar mice fed a diet deficient in vitamin A, following a single topical dose of dimethylbenzanthracene. The differences were due at least in part to more rapid loss of papillomas in the vitamin-A-supplemented animals. Within a single dietary group the rate of loss was linearly dependent on the total number of papillomas produced, but this rate was substantially higher in supplemented animals. Both appearance and loss of papillomas were linear functions of time, in supplemented animals, whereas the rates in nonsupplemented animals appeared to be of higher orders.

N-Benzoyloxy-N-methyl-4-aminoazobenzene: Its Carcinogenic Activity in the Rat and Its Reactions with Proteins and Nucleic Acids and Their Constituents in Vitro

Abstract: LIONELA.POIRIER,-JAMESA.MILLER,ELIZABETHCMILLER,ANDKEISATOMcArdleLaboratoryforCancerResearch,MedicalCenter,UniversityofWisconsin,Madison,Wisconsin53706SUMMARYAT-Benzoyloxy-A'-methyl-4-aminoazobenzene(.V-benzoyloxy-MAB),anewderivativeofthehepatocarcinogenAr-methyl-4-aminoazobenzene(MAAŸ),wassynthesized.ThistoxicesterofA'-hydroxy-MABproducedsarcomasatthesiteofrepeateds.c.injectionsinrats;MABwasinactiveundertheseconditions.UnlikeMAB,Ar-benzoyloxy-MABreactedreadilyinvitroatpH7withprotein,RNA,andDXAtoformmacromolecular-bounddye.Fivenucleophiliccomponentsofthesemacro-molecules(methionine,cysteine,tryptophan,tyrosine,andguanosine)reactedwithAT-benzoyloxy-MABundersimilarconditionstoformpolardyes;othercommonaminoacidandnucleosidecomponentsdidnotreact.ThereactionsofN-benzoyloxy-MABwithproteins,nucleicacids,andtheircomponentsmaybeusefulprototypesinstudiesontheprotein-andnucleicacid-bounddyesformedinvivobytheaminoazodyes.ThebiologicandchemicalpropertiesofAr-benzoyloxy-MABparallelthoseofthecarcinogenicaromaticN-acyloxyamideAr-acetoxy-2-acetylaminofluoreneandlendfurthersupporttotheconceptthatthecarcinogenicaromaticaminesandamidesareactivatedinvivobyesterificationoftheirA^-hy-droxymetabolites.INTRODUCTIONAr-Hydroxylationhasbeendemonstratedasanimportantstepinthemetabolicactivationofcarcinogenicamides.Thus,AAF4(9,27)andits7-fluoroderivative(20),4-acetylamino-1Thisinvestigation wassupported byResearchTrainingGrantCRTY-5002andbyProgram-Project GrantCA-07175oftheNationalCancerInstitute, USPHS;byagrantfromTheJaneCoffinChildsMemorialFvmdforMedicalResearch;andbytheAlexanderandMargaretStewartTrustFund.2MontrealCancerInstitute,NotreDameHospital,Montreal,Quebec,Canada.3Department ofEducation, HirosakiUniversity, Hirosaki,Aomori-ken,Japan.4Thefollowingabbreviationsareused(theChemicalAbstractsnomenclatureisgiveninparentheses): AB,4-aminoazobenzene(p-phenylazoaniline); AAB,A7-acetyl-4-aminoazobenzene (p-phenylazoacetanilide); MAB, Ar-methyl-4-aminoazobenzeneOV-methyl-p-phenylazoaniline); DAB,JV,AT-dimethyl-4-aminoazobenzene(Ar,Ar-dimethyl-p-phenylazoaniline); AAF,2-acetyl-aminofluorene(A^-fluorenylacetamide).ReceivedMarch27,1967;acceptedMay6,1967.biphenyl(35),4-acetylaminostilbene(2,4),and2-acetyl-aminophenanthrene(20)areeachmetabolizedtoA^-hydroxyderivativesbyspeciesinwhichtheyarecarcinogenic.Furthermore,whenadministeredtorats,theseW-hydroxymetabolitesaremorecarcinogenicthantheparentcompoundsatsiteswheretheamidesareactiveandproducetumorsatsitesoflocalapplicationwheretheparentcompoundshavelittleornoactivity(2,5,20,23,24,28,35,39).TheisolationofN-hydroxy-AT-acetyl-4-aminoazobenzenefromtheurineofrats,mice,orhamstersadministeredABoritscarcinogenicA'-methylorA*,A-dimethylderivatives(MABorDAB)showedthattheaminoazodyesarelikewiseAr-hydroxylatedinvivo(42).However,neitherthismetabolitenorA^-hydroxy-ABwascarcinogeniconadministrationtorats(42).TheinactivityofthesecompoundsisconsistentwiththeapparentrequirementofaA'-methylgroupforstrongcarcinogenicityintheseriesofaminoazodyesstructurallyrelatedtoMABandDAB(3,6,29).Thesedatasuggestedthattheproximatecarcinogenicmetabolite(s)ofthesedyeswouldcontainbothaA^-methylgroupandaA"-hydroxygroup.WhenrepeatedattemptstosynthesizeA'-hydroxy-Af-methyl-4-aminoazobenzene(JV-hydroJSy-MAB)failed,itsO-benzoylderivative,A"-benzoyloxy-MAB,waspreparedonthepremisethatitmightreleasethepresumedcarcinogenicmetaboliteinvivo.Thisesterinducedahighincidenceofsarcomasinratsonrepeatedsubcutaneousinjection.Furthermore,A'-benzo-yloxy-MABreactednonenzymaticallyinneutralsolutionwithcertainaminoacidsorproteinsandwithguanosine,DXA,andRNA.Thesedata,whichformthesubjectofthisreport,andtherecentstudiesfromthislaboratoryonthesimilarreactivityandcarcinogenicityofAr-acetoxy-AAF(10,14-16,19)supporttheconcejilthatA"-hydroxymetabolitesofcarcinogenicaromaticaminesandamidesareactivatedforcarcinogenesisbyesterificationoftheAT-hydroxygroup(32).MATERIALSANDMETHODSPreparationofCompoundsSynthesisofN-Beiizoyloxy-MAB.A^-Benzoyloxy-MABwassynthesizedbyamodificationoftheprocedureusedbyHornerandSteppan(13)forthesynthesisofAr-benzoyloxy-A"-ethylaniline.Itwasfoundnecessarytoperformalloperationsbetween—2°Cand5°C,andallsolutionswerealsomaintainedwithinthistemperaturerange.MAB(20gm)dissolvedin70mlKXX) CANCERRESEARCHVOL.27