Inhibition of dimethylnitrosamine-induced strand breaks in liver DNA and liver cell necrosis by diethyldithiocarbamate.

Diethyldithiocarbamate (DEDTC) prevented dimethylnitrosamine (DMN)-induced strand breaks in liver DNA and liver cell necrosis in male Wistar rats. In contrast, DEDTC did not inhibit the fragmentation of liver DNA caused by several other chemical carcinogens (N-hydroxy-2-acetylaminofluorene, 3-hydroxyxanthine, aflatoxin B1, N-acetoxy-2-acetylaminofluorene, methyl methanesulfonate, methylnitrosourea, and methylazoxy-methanol acetate), whether or not they required metabolic activation. Aminoacetonitrile exerted an action similar to that of DEDTC. The inhibitory effect was transitory, lasting at least for 4 hours, and protection for longer than 4 hours required multiple administrations of DEDTC. DEDTC also inhibited the serum clearance of DMN, methylation of liver DNA, and oxidative demethylation of DMN in the in vitro hepatic microsomal system prepared from either male Wistar rats or from hamsters. Interference of the metabolism of DMN appeared to be the mechanism by which DEDTC arrested DMN-induced biochemical and biologic effects.

Effect of combined tigason and selenium treatment on colon carcinogenesis.

The modifying effect of an aromatic retinoic acid analog. Tigason, alone or in a combined regimen with selenium on the incidence of colon tumors induced by 1,2-dimethylhydrazine was investigated in BD rats. Treatment with the carcinogen alone resulted in induction of colon adenocarcinomas in 29 rats (92%). In comparison to the 1,2-dimethylhydrazine-treated group, administration of Tigason or Tigason plus selenium reduced tumor incidence (71% and 68%, respectively). The effect of combined treatment with Tigason and selenium, however, was not additive. Although some researchers have been unable to demonstrate any protective effect of different synthetic retinoids against 1,2-dimethylhydrazine or N-methyl-N-nitrosourea-induced colon tumors, our findings suggest that the process of tumor initiation in colon mucosa can be significantly influenced by the aromatic retinoid Tigason or by simultaneous administration of Tigason and selenium.

The soft-tissue tumours induced in Syrian hamsters by herpes simplex virus type 1 and a chemical promoter.

Adult male Syrian hamsters were inoculated subcutaneously with herpes simplex virus type 1 (HSV-1, 10(6) PFU) or ultraviolet-inactivated HSV-1. One week later 12-O-tetradecanoylphorbol 13-acetate (TPA, 2 x 20 nmol weekly) was topically applied to the dorsal skin at the site of virus inoculation for 6 months. Control animals received HSV-1 only or topical treatment with TPA in acetone or acetone alone. Small tumour nodules developed in the HSV-1 group close to the site of virus inoculation 10-15 months after the beginning of the experiment. The neoplasms were classified as angiolipomas, chondromyxomas, a hibernoma, and an unclassified tumor resembling a Kaposi sarcoma in humans. The topical TPA treatment alone induced melanocytic hyperplasia and sebaceous gland hyperplasia. The soft-tissue tumours differed markedly from the structure of the soft-tissue sarcomas induced in Syrian hamsters by viruses of the papova and polyoma groups. Since the spontaneous incidence of benign soft-tissue tumours in our close hamster colony is extremely low, we concluded that mutagenic HSV-1 effects on hamster mesenchymal cell DNA may be involved in the process of formation of the observed benign neoplasms.

Effect of disulfiram on the alkylation of rat liver DNA by nitrosodiethylamine.

The extent of ethylation of guanine in 7 N and O 6 position in rat liver DNA was studied after chronic feeding with unlabelled nitrosodiethylamine followed by a single application of the 14C labelled drug. Disulfiram inhibits the alkylation of rat liver DNA. It was also shown that a discontinuous dosage of disulfiram protects against the alkylating effect of 14C nitrosodiethylamine only for a distinct period of time.

Proline dithiocarbamate inhibits N-nitrosodiethylamine induced liver carcinogenesis.

A study was conducted to determine the toxicity of different dithiocarbamates and of disulfiram. In an experiment showing the cytotoxicity against murine spleen lymphocytes, proline dithiocarbamate (PDTC) and thioproline dithiocarbamate showed the lowest toxicity. Therefore one of them was selected and different doses of the hydrophilic PDTC were checked for their ability to affect the development of liver and oesophagus tumours induced in BD-6 rats by N-nitrosodiethylamine (NDEA). Rats were injected i.p. with 80 mg/kg NDEA once weekly for 10 weeks. Administration of PDTC, 1 h before and 24 h after the carcinogen, markedly decreased the number of rats developing NDEA-induced hepatocellular carcinoma and liver haemangioendothelioma. A 59%-77% reduction in the incidence of liver tumours was found in the different groups when the carcinogen was administered in combination with the inhibitor. For least 40 weeks after the start of the experiment PDTC protected the liver from NDEA carcinogenesis and did not shift the tumour development to any other organ. PDTC did not significantly affect the weight gain of the experimental animals. We conclude that parenteral administration of PDTC seems to represent a promising approach in chemoprevention of liver carcinogenesis.

Effects of disulfiram on the metabolism of nitrosodiethylamine during liver carcinogenesis.

We have studied the effects of disulfiram (DSF) administration on the metabolism of nitrosodiethylamine (NDEA) in rats during acute and chronic administration. DSF was found to have the following effects during the course of carcinogenesis: (a) marked decrease in the exhalation of 14CO2 derived from 14C-NDEA; (b) reduction of the total levels of DNA and RNA ethylation in the liver. In acute experiments DSF caused an increase in the amount of NDEA in organs and in the urine. We suggest that inhibition of NDEA biotransformation and the subsequent decrease in the total level of DNA ethylation may prevent specific chemical interactions relevant to carcinogenesis.

Non-random distribution of N-methyl-N-nitrosourea sensitive sites in a eukaryotic genome.

DNA released from Ehrlich ascites cells by lysis in the presence of 50 microgram X ml-1 of proteinase K contains long alkali-stable strands in the order of 50-100 X 10(6) daltons. In contrast, DNA released in the presence of 6 mg X ml-1 of autodigested pronase is significantly nicked. According to sedimentation rates the number of internal ends liberated during this procedure is 24/200 X 10(6) daltons. The number of alkali-labile sites introduced into DNA by incubation of Ehrlich ascites cells with 1 nM of N-methyl-N-nitrosourea (MNU) followed by cell lysis in the presence of 50 microgram X ml-1 of proteinase K and alkali-denaturation is 16.6/200 X 10(6) daltons. From this one should expect that denatured DNA released from cells pretreated with 1 mM of MNU which are subsequently lysed with 6 mg X ml-1 of pronase would have about 40 single-strand breaks/200 X 10(6) daltons. However, denatured DNA strands released by 6 mg X ml-1 of pronase either from MNU-treated or untreated cells cannot be separated by centrifugation through alkaline sucrose gradients. This phenomenon could be explained by a non-random distribution of MNU-inducible alkali-labile sites of DNA in vivo.

Comparative pharmacokinetic and photodynamic studies with zinc(II) phthalocyanine in hamsters bearing an induced or transplanted rhabdomyosarcoma.

Comparative pharmacokinetic studies in hamsters bearing an induced or first-generation transplanted rhabdomyosarcoma that were injected with liposome-incorporated zinc(II) phthalocyanine (ZnPc) show a higher drug concentration in the induced tumour. The selectivity of tumour targeting is underlined by the fact that, 24 h after injection, larger amounts of ZnPc are found in the tumour than in the liver. Photodynamic therapy investigations were carried out using 673 nm light from an argon-dye laser. On the basis of different assessment criteria (changes in mean tumour diameter with time, tumour mass regression, survival time of the treated groups of animals, and histological determination of the necrotic tissue) the photosensitizing effect of ZnPc appears to be comparable for both kinds of tumour in spite of the higher uptake of photosensitizer by the induced tumour.

Strand breakage in rat brain DNA and its repair induced by ethylnitrosourea in vivo.

The damage and repair of rat brain DNA was studied in vivo after a single carcinogenic dose of ethylnitrosourea. Fragmentation of the brain DNA produced by this carcinogen was demonstrated on alkaline sucrose gradients. By the 24th hrs after treatment with ethylnitrosourea the single-strand damage to DNA was not completely repaired. As the highly differentiated cells of the central nervous system do not proliferate, it is possible that during brain carcinogenesis delayed repair of DNA of primitive cells might be needed for the formation of tumor anlage.

Chromosomal proteins in hepatocarcinogenesis.

The chromosomal proteins of rat liver were studied by SDS-gel electrophoresis during the process of nitrosomorpholine-induced hepatocarcinogenesis, in the primary hepatomas thus obtained, and in their metastases. It was found that an increased proteolytic activity was present in liver homogenates from carcinogen-fed animals which caused differences between the nonhistone chromosomal proteins of control and carcinogen-treated livers. These differences disappeared in the presence of the protease inhibitor PMSF. In the primary hepatomas slight quantative changes were observed: an increased amount of two proteins of 43000 and 63000 daltons molecular weight, respectively, and a decrease in the histone subfraction H 1 degrees. In the metastases both quantative and qualitative differences were detected: a strong decrease in the protein bands corresponding to the contractile proteins alpha-tubulin, beta-tubulin, and actin; an increased content of the 63000 dalton protein; the appearance of new proteins of approximately 60000, 90000, and 120000 daltons molecular weight, and the complete disappearance of histone H 1 degrees.

Differentiation of malignant myoblasts in 7,12-dimethyl-benz(a)-anthracene-induced rhabdomyoblastomas. An electron microscopic study.

The differentiation of DMBA-induced rhabdomyoblastomas was studied during the early stages of tumour growth. Two cell populations were found to constitute the tumour tissue: small cells (SC) and long spindle-shaped cells (LSSC). The SC were the only tumor cells at the earliest detectable stage of tumour growth 10 weeks after intramuscular injection of DMBA. They had small heterochromatic nuclei with a compact nucleolus containing only fibrillar components. The cytoplasm was very rich in SER of tubular type, dense bodies and Golgi apparatus. Centrioles at all stages of the replicative cycle were very frequently observed. The cells did not fuse and showed no tendency to differentiate. The LSSC had large euchromatic nuclei with multiple irregular nucleoli containing both fibrillar and granular components. The cytoplasm had an abundant GER and well-developed Golgi apparatus. These cells formed 100 Angstrom thick cytofilaments the increase of which paralleled reduction of GER. The cells tended to fuse but did not form myofibrils. A rare variant of these cells neither possessed Golgi apparatus nor formed cytofilaments but accumulated dense protein substance in the cisternae of the GER. Myotubes with cross-striated myofibrils were but occasionally observed. The ultrastructural characteristics of both cell types revealed essential differences in the biosynthetic activity and the degree of differentiation. The SC were considered to belong to the myogenic cell line and to be most probably the malignant counterpart of proliferating satellite cells (presumptive myoblasts) and precursors of the LSSC. Morphologically and developmentally the LSSC bore close resemblance to normal myoblasts but the proliferative capacity of some of them seemed to be lost. The differentiation of the malignant myoblasts in the DMBA-induced rhabdomyoblastomas was similar to the early differentiation of the normal muscle tissue.

Inhibition of diethylnitrosamine-induced strand breaks in liver DNA by disulfiram.

Disulfiram (DSF) delayed the appearance of diethylnitrosamine (DEN)-induced strand breaks in liver DNA of rats. The fragmentation of liver DNA produced by DEN was studied 4 and 24 h after administration of the carcinogen on alkaline sucrose gradients. A single dose of 500 mg/kg DSF given 1 h prior to carcinogen treatment delayed for at least 4 h the DEN--induced strand breaks in liver DNA. A single DSF pretreatment, however, did not protect against carcinogen-induced strand breaks when observed 24 h after DEN injection. It is possible that continuous administration of DSF might inhibit the DEN-produced damage of the genetic material.

Influence of disulfiram on the metabolism of N-nitrosodiethylamine.

The effect of disulfiram (DSF) on the biological activity of N-nitrosodiethylamine (NDEA) was studied in vivo. We determined its influence on NDEA level, on NDEA-induced DNA damage and on DNA alkylation in the liver and oesophagus. It was found that 500 mg/kg DSF given 2 h before a single dose of 28 mg/kg NDEA inhibited the metabolism and increased the concentrations of NDEA in the organs, especially in the oesophagus. Consequently, DNA damage and the alkylation of DNA are inhibited in both the liver and the oesophagus.

Effects of potassium ethylxanthogenate on nitrosodiethylamine-induced DNA damage and on liver carcinogenesis.

BD-6 rats were injected with 80 mg/kg N-nitroso-diethylamine weekly for 10 weeks. Addition of 270 mg/kg potassium ethylxanthogenate weekly reduced significantly the number of rats developing NDEA-induced malignant liver tumors. Ethylxanthogenate decreased the total number of liver tumors induced by the carcinogen to 6 as compared to a total of 29 neoplasms in animals treated only with NDEA. In acute experiments potassium ethylxanthogenate markedly decreased the exhalation of 14CO2 derived from 14C-NDEA. The amount of the nonmetabolized carcinogen increased in the urine of ethylxanthogenate protected rats only 4% of the given dose. Initial DNA damage, single strand breaks and alkali-labile sites, was determined by alkaline sucrose gradients and in protected animals was minimal for at least 24 hours after NDEA administration. It appears that the production of less initial DNA damage may be important for the further course of liver carcinogenesis induced by relatively large doses of nitrosodiethylamine.

Effect of diallyl sulfide on aristolochic acid-induced forestomach carcinogenesis in rats.

In this study the development of aristolochic acid (AA) induced tumors in rats with and without diallyl sulfide (DAS) was studied. Experiments were also conducted to establish the effects of DAS administration on AA-derived DNA single-stranded regions and DNA adduct formation in the forestomach of such animals. Forestomach, urinary bladder and thymus tumors were induced in male BD-6 rats after oral treatment for 12 weeks with AA (2 x 10 mg/kg/week). Administration of 150 mg/kg DAS intragastrically 4 h prior to AA treatment reduced significantly the number of rats that developed forestomach tumors (6-9 months after the start of experiment). The incidence of AA-induced forestomach tumors was 10% (two out of 20 rats) after co-administration of DAS and 60% (12 out of 20 animals) when AA was administered alone. The high dose of DAS (2 x 150 mg/kg) markedly inhibited the formation of squamous cell carcinomas in the forestomach. However, the thioether did not prevent the formation of forestomach and urinary bladder papillomatosis. Additionally, DAS co-administration decreased the accumulation of single-stranded regions in rat forestomach DNA. Using the nuclease P1 enhancement method of the 32P-postlabeling assay, a decrease in the level of AA-derived adducts was also detected after co-administration of DAS. We conclude that the decrease of DNA damage after DAS co-administration is associated with the delay in conversion of papillomas to malignant forestomach tumors.