Liver cancer: neonatal estrogen enhances induction by a carcinogen.

A single injection of 100 micrograms of estradiol benzoate into newborn rats was followed after weaning by dietary treatment with one of two dosages of the carcinogen N-hydroxy-N-2-fluorenylacetamide. Autopsies 26 weeks later showed a higher incidence of liver cancer in male and, particularly, female rats injected with hormone than in controls. The weights of livers were greater but gonads were smaller in size in the estradiol groups. Endocrine and possibly centralnervous-system factors may play roles in formation of liver tumors.

Effect of chlorpromazine hydrochloride on carcinogen-metabolizing enzymes: liver microsomal dimethylnitrosamine demethylase, 4-dimethylaminoazobenzene reductase, and aryl hydrocarbon hydroxylase.

Investigation of the effect of chlorpromazine hydrochloride (CPZ) on some enzymes involved in the metabolism of the carcinogens 4-dimethylaminoazobenzene (DAB), benzo[a]pyrene, and dimethylnitrosamine (DMN) revealed that CPZ inhibited hepatic microsomal DAB reductase, aryl hydrocarbon hydroxylase, and DMN demethylase II but markedly activated DMN demethylase I. Inhibition of these enzymes in vitro was proportional to the concentrations of CPZ. The effect of CPZ on DMN demethylase also depended on the concentrations of DMN and CPZ in the incubation mixture. Mechanisms to account for the inhibition of DAB reductase were suggested. Aryl hydrocarbon hydroxylase and DMN demethylase II activities of the 100,000 x g hepatic microsomal fraction were activated by 33 and 61%, respectively, over the control values after a single injection of CPZ into F344 rats, but no effect on DAB reductase and DMN demethylase I activities was observed. Microsomal concentrations of protein and cytochrome P450 were not appreciably altered by CPZ treatment, whereas the level of microsomal NADPH cytochrome c reductase was slightly increased over control values. A similar effect on the drug-metabolizing enzymes was found during pretreatment of rats with CPZ for 5 days, except that the NADPH cytochrome c reductase was increased by 33% over the control values.

Selection of carcinogens and related compounds tested for mutagenic activity.

A list of 102 chemicals was prepared for subsequent mutagenesis assays in a National Cancer Institute program to determine the extent of correlation between carcinogenesis and mutagenesis in standardized assays. The chemicals were divided into five major categories: 37 aromatic amines, 11 polycyclic aromatic hydrocarbons, 8 nitrosamines and nitrosamides, 16 alkylating agents, and a miscellaneous category consisting of 11 heterocyclic compounds, 7 amides, ureas and acylating agents, 5 antimetabolites, 4 inorganic chemicals, and 3 promoters. The chemicals were further described as procarcinogens (requiring metabolic activation to exert their biologic activities), ultimate carcinogens (direct-acting chemicals not requiring metabolic activation), and noncarcinogens (compounds shown to be inactive in one or more adequate carcinogenicity tests). An extensive bibliography documents the selection and categorization of the compounds.

Mutagenic activation of N-2-fluorenylacetamide and N-hydroxy-N-2-fluorenylacetamide in subcellular fractions from X/Gf mice.

The Salmonella mutagenesis test system was used to evaluate the in vitro mutagenic potency of N-2-fluorenylacetamide (2-FAA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) mediated by liver and kidney subcellular fractions from X/Gf mice, a strain resistant to 2-FAA carcinogenesis. Pretreatment of the mice with the microsomal inducers 3-methylcholanthrene (MCA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased the number of revertants from both liver and kidney fractions. Mutagenicity of N-OH-2-FAA mediated by liver or kidney microsomes was partially inhibited at 0.001--0.1 microM Paraxon (diethyl-p-nitrophenyl phosphate), an inhibitor of deacetylase enzyme, and the inhibition was complete (98%) in microsomes from control mice (100 microM Paraoxon). Conversely, the liver and kidney microsomal fractions from MCA- and TCDD-treated X/Gf mice were less sensitive to Paraoxon. The inhibition of kidney or liver cytosol-mediated N-OH-2-FAA mutagenicity by Paraoxon was less than that observed with the microsomal fraction (50% inhibition at 1 x 10(-7) and 1 x 10(-5) M Paraoxon, respectively). The mutagenicity of 2-FAA and N-OH-2-FAA mediated by liver or kidney subcellular fractions from X/Gf mice and its response to inducers and inhibitors of mutagenic activation processes appear similar to those observed in species both resistant (cotton rat) and sensitive (Sprague-Dawley rat, NIH Swiss mice) to 2-FAA carcinogenesis.

Metabolism of N-2-fluorenylacetamide in X/Gf mice: lack of correlation between biochemical interaction and carcinogenicity.

A comparative study of the metabolism of the carcinogen N-2-fluorenylacetamide by mice of the X/Gf strain (resistant to the tumorigenic action) and the NiH Swiss strain (susceptible to the tumorigenic action) showed slight but not outstanding differences in metabolic patterns. The activated metabolite N-hydroxy-N-2-fluorenylacetamide was excreted by both strains, and the levels of carcinogens or metabolites bound to liver macromolecular constituents were comparable in the X/Gf and NiH Swiss mice.

Tumor response in strain A mice exposed to silylating compounds used for gas-liquid chromatography.

Six reagents used to silylate, alkylate, or acylate compounds for ease of identification on gas chromatographic columns significantly increased the frequency of lung tumors in A/He mice.

Pulmonary adenoma response of strain A mice to sulfonic acid derivatives of 1- and 2-naphthylamines.

Various sulfonic acid derivatives of 1-naphthylamine and 2-naphthylamine were tested in inbred A/St (male and female) mice by the pulmonary adenoma bioassay to determine if this class of compounds, used as intermediates in the dye-stuff industry, possesses tumorigenic activity. Neither 1-naphthylamine nor the four sulfonic acid derivatives of 1-naphthylamine tested were tumorigenic. However, 2-naphthylamine and two of the three sulfonic acid derivatives of 2-naphthylamine tested produced statistically significant lung tumor responses at comparable doses. These results indicated that this class of compounds should be examined more extensively for carcinogenic activity.

Effect of lifetime exposure to aflatoxin b1 in rats.

Aflatoxin B1 was fed at 2 ppm in the diet to a group of pregnant F344 rats from the time of conception; it was then fed to their offspring until death. This diet was also given to another group of rats 6-7 weeks old for comparison. The survival time of male rats was significantly shorter than that of the female rats of both groups. However, the survival times of rats of the same sex in both groups did not differ significantly. The major causes of death were hepatic neoplasms with matastases, although some early deaths occurred before neoplasms developed. Most deaths were from a malignant hemorrhagic liver tumor, histologically diagnosed as a hemangiosarcoma, which caused rupture and hemorrhage into the peritoneal cavity or metastases to the lungs. These hemangiosarcomas were readily transplantable and did not produce alpha-fetoprotein. Ultrastructurally, they were composed of poorly differentiated cells resembling endothelial cells. Nodules of hyperplasia induced by aflatoxin B1 sometimes grew large (greater than 1.5 cm), and 2 were transplanted. Approximately 20% of the rats had colon tumors; a few rats had tumors of the kidney, oral cavity, and hematopoietic system.

Neoplastic response of F344 rats and B6C3F1 mice to the polymer and dyestuff intermediates 4,4'-methylenebis(N,N-dimethyl)-benzenamine, 4,4'-oxydianiline, and 4,4'-methylenedianiline.

Feeding 4,4'-methylenebis(N,N-dimethyl)benzenamine [CAS: 101-61-1; 4,4'-methylenebis(N,N-dimethylaniline)] to inbred F344 rats increased the incidence of thyroid lesions (hyperplasia, adenomas, and carcinomas) in both sexes, especially in the female rats. 4,4'- Oxydianiline (CAS: 101-80-4) in the diet increased adenomas and carcinomas in the thyroid gland and neoplastic nodules and carcinomas in the liver of male and female F344 rats. In addition to increasing thyroid adenomas in females and hepatocellular adenomas or carcinomas in male and female B6C3F1 mice, 4,4'- oxydianiline increased adenomas of the harderian gland in male and female mice. 4,4'- Methylenedianiline (CAS: 101-77-9) in the drinking water increased neoplasms of the thyroid gland and liver in F344 rats and B6C3F1 mice.

Carcinogenicity of three dose levels of 1,4-Bis(4-fluorophenyl)-2-propynyl-N-cyclooctyl carbamate in male Sprague-Dawley and F344 rats.

Feeding 1,4-bis(fluorophenyl)-2-propynyl-N-cyclooctyl carbamate to F344 or Sprague-Dawley male rats at 125-500 ppm in the diet led to many carcinomas of the small intestine and ear duct (Zymbal's gland tumors) in addition to lymphomas and leukemias. A high incidence of mammary adenocarcinomas was found in the Sprague-Dawley rats. Only a few colon or liver tumors were observed in each strain.

Effect of microsomal enzyme inducers on the urinary excretion pattern of mutagenic metabolites of the carcinogen 2,4-toluenediamine.

2,4-Toluenediamine [(TDA) CAS: 95-80-7] was administered to rats pretreated with the microsomal enzyme inducers phenobarbital (PB), beta-naphthoflavone (beta NF), or 3-methylcholanthrene (MCA). The 24-hour urines of male F344 rats were examined for their mutagenic potency by means of the Salmonella assay, with the Aroclor 1254-pretreated rat liver S-9 fraction as an activating system. No revertants were found with TDA or its urinary metabolites in the absence of the S-9 fraction. In the presence of S-9, the number of revertants increased as the concentration of TDA or its urinary metabolites increased. The urinary metabolites, generated after the microsomal enzyme inducers (PB, beta NF, MCA), had increased mutagenic activity as compared with the controls (saline, corn oil). In the presence of beta-glucuronidase (beta G), increased numbers of TA98 revertants were noted in the urine of rats pretreated with PB, saline, or corn oil. Addition of sulfatase did not alter the number of TA98 revertants. Conversely, beta G treatment of urine from rats pretreated with MCA or beta NF led to a decrease in the number of TA98 revertants as compared to levels in urine without beta G. Addition of known urinary metabolites of TDA, such as 4-acetylamino-2-aminobenzoic acid or 2,4-diacetylaminobenzoic acid, to beta NF-pretreated rat urine had no inhibitory effect on the mutagenicity in the absence of beta G. However, in the presence of beta G, the inhibitory effect was similar to that noted with beta NF-pretreated rat urine. Upon separation of urinary metabolites (beta NF-pretreated rat urine) into free, conjugated, and water-soluble forms, the maximum number of TA98 revertants was associated with the free ethyl acetate-extractable fraction, which accounted for the total mutagenic activity associated with the original volume of urine. Conjugated metabolites showed much less mutagenic activity, and an inhibitory principle was associated with the water-soluble fraction.

A carcinogenicity assay of Mirex in Charles River CD rats.

The long-term administration of 50 and 100 ppm of Mirex in the diets of male and female Charles River CD rats was associated with a spectrum of liver lesions, from foci or areas of cellular alteration and neoplastic nodules to hepatocellular carcinoma. Statistically significant numbers of neoplastic nodules were observed in the livers of male rats receiving the high dose. Neoplastic nodules and hepatocellular carcinomas were not observed in control rats.

Mechanisms of the inhibitory action of p-hydroxyacetanilide on carcinogenesis by N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide.

The metabolism of N-2-fluorenylacetamide (FAA) and N-hydroxy-2-fluorenylacetamide (N-OH-FAA) was studied in groups of rats that had been prefed the protective agent p-hydroxyacetanilide (p-OH-AA) alone or in combination with each of the carcinogens for 4 weeks. Compared with controls, pretreatment increased the percentage of metabolites in the urine, chiefly as glucuronic acid conjugates, whereas the fecal excretion of FAA metabolites was lowered. The levels of total and tissue-bound material in the liver and blood plasma were also lower after prefeeding. Liver aryl hydrocarbon hydroxylase and liver deacetylase were not affected by p-OH-AA pretreatment. However, liver glucuronyl transferase was increased by either prefeeding with p-OH-AA and/or the carcinogen. The protective effect of p-OH-AA against liver tumor induction with FAA or N-OH-FAA may in part result from a combination of the decreased binding of carcinogen to hepatic cellular macromolecules and the increased excretion as the glucuronide conjugates.

Aging changes in CD-1 HaM/ICR mice reared under standard laboratory conditions.

Three hundred CD-1 HaM/ICR mice were observed for 2 years, and useful necropsies were done on 99 males and 102 females. Mortality was 50% at 16 months in the males and 18 months in the females. Among mice that came to autopsy, total tumor incidence was 54% for males and 75% for females, with most neoplasms occurring after 18 months. Adenomas or adenocarcinomas of the lung were the most frequent, followed by lymphoreticular tumors, vascular tumors, hepatomas, subcutaneous fibrosarcomas, and adenocarcinomas of the mammary glands. Some degree of amyloidosis was seen in half the mice of both sexes, beginning at 8 months in males and 12 months in females. Variability in tumor incidence among small groups if mice emphasized the need for adequate samples.

Modification of diethylnitrosamine liver carcinogenesis with phenobarbital but not with immunosuppression.

Administration of 40 ppm diethylnitrosamine (DENA) in the drinking water for 10 weeks to male Fischer rats led to hepatocellular carcinoma in 100 percent with metastasis to the lung in 40 percent, of the animals living for the full experimental period of 20 weeks. Concurrent feeding of phenobarbital and DENA for 10 weeks produced cancer of the liver in 77 percent of the animals, but only 9 percent had metastases in the lung. A brief regimen of DENA for 4 weeks, followed by 16 weeks of observation, induced cancer of the liver in only 13 percent of the rats. Administration of phenobarbital, begun 1 week after cessation of DENA intake and terminated at week 20, led to liver cancer in 64 percent of the rodents. Hydroxyurea had no effect on this enhancement. Treatment with a purified gamma fraction of antilymphocytic serum after the DENA did not influence the outcome. Thus phenobarbital given together with DENA reduced the severity of the carcinogenic process, but when it was given after the hepatocarcinogen, it increased the effect.

Carcinogenicity tests of certain environmental and industrial chemicals.

Fourteen chemicals of varied uses were tested for carcinogenicity by oral administration in male and female Charles River CD rats. Under the conditions of the tests, propane sultone, propylene imine, and ethylenethiourea, in addition to the positive control N-2-fluorenylacetamide, were carcinogenic. Avadex, bis(2-chloroethyl) ether, the potassium salt of bis(2-hydroxyethyl) dithiocarbamic acid, ethylene carbonate, and semicarbazide hydrochloride were not carcinogenic under the test conditions. Dithiooxamide, glycerol alpha-monochlorohydrin, and thiosemicarbazide gave somewhat ambiguous results, though administered at high enough dose levels to be toxic. An inadequate number of animals survived treatments with sodium azide, sodium bisulfide, and vinylene carbonate, or the animals may not have received sufficiently high doses of the test chemicals to provide maximum test sensitivity. However, there were no indications that these three chemicals were carcinogenic under the test conditions.

Transformation of human osteosarcoma cells by a chemical carcinogen.

A human osteosarcoma clonal cell line (TE-85, clone F-5) was treated in vitro with various levels of 7,12-dimethylbenz[a]anthracene or dimethyl sulfoxide (control). Cells treated only with the carcinogen underwent morphologic alteration in vitro, and one of these altered cell lines produced tumors subcutaneously and intracerebrally when injected into NIH nude mice.

Techniques for carcinogenicity studies.

Short-term tests to detect genetic, chromosomal, or DNA damage are now required by regulatory agencies for any new compound proposed for commercial production. Furthermore, full-scale carcinogenicity tests may be required for certain compounds. In this circumstance, the compound-related factors including stability, purity, physical properties, and chemical structure and reactivity must be considered. Animal factors include species and strain of test animal, route of administration, age, sex, diet, and spontaneous tumor incidence. A team of qualified investigators with experience in various disciplines is required to conduct the studies properly. Quality control measures and adherence to the code of good laboratory practice are also necessary during all phases of the study. The investment in a carcinogenicity study therefore becomes fairly substantial in terms of both time and money.

Intestinal tumors in mice treated with a single injection of N-nitroso-N-butylurea.

N-Nitroso-N-butylurea was injected once at dosage levels of 150 or 75 mg/kg into 3- or 6-week-old male C57BL/L mice. Intestinal tumors occurred in 100% of the mice that survived more than 15 weeks after injection with a high dose of N-nitroso-N-butylurea at 6 weeks of age, and in 35 to 70% of the mice in other treatment groups. These intestinal tumors were seen primarily at the junction of the pylorus and duodenum and in the anterior portion of the small intestine, with a few in the cecum, colon, and rectum. The tumors at the junction were not very invasive tumors and frequently appeared as polypoid growths. Tumors not at this location were adenocarcinomas that invaded all the layers of the gut wall but which did not metastasize. Colorectal tumors were adenomas and adenocarcinomas. N-Nitroso-N-butylurea also induced tumors of the stomach, hematopoietic system, lung, and liver.

Test for carcinogenicity of organic contaminants of United States drinking waters by pulmonary tumor response in strain A mice.

The production of lung adenomas in strain A following multiple i.p. injections of selected organic water contaminants was investigated. Of the 16 contaminants tested, only bromoform produced a pulmonary adenoma response that was significantly greater than the pulmonary adenoma response of vehicle-treated control mice.