Showing papers in "Carcinogenesis in 1981"

Diminution of mouse epidermal superoxide dismutase and catalase activities by tumor promoters

Abstract: The effects of phorbol ester tumor promoters and related compounds on superoxide dismutase (SOD) and catalase were examined. The treatment of adult mouse skin with 2 ..mu..g 12-0-tetradecanoylphorbol-13-acetate (TPA) resulted in a sustained decrease in the basal levels of both SOD and catalase activities in the epidermis. A decline in SOD activity occurred within 2 h after application and the maximum effect was seen at 16-17 h. The decrease in SOD activity was always accompanied by a similar decline in the epidermal catalase activity. The alterations in both enzymes occurred against a high background of enhanced protein synthesis which indicates that the effect of TPA is selective for SOD and catalase. Other tumor promoters such as phorbol 12,13-dibutyrate and the non-phorbol tumor promoter anthraline also lowered the activities of both the enzymes. Mezerein, a resiniferonol derivative with weak promoting activity but a potent stage-II promoter, appeared to be more potent than TPA in lowering the basal levels. These results indicate that damage which favors neoplastic progression would occur in TPA-treated mouse skin due to the accumulation of free radicals resulting from low levels of SOD and catalase activity. In addition, the TPA-caused decrease in the levels of SOD and catalasemore » was not prevented by either retinoic acid, fluocinolone acetonide, tosyl amino-2-phenylethyl chloromethyl ketone, or butylated hydroxytoluene, suggesting that inhibition of tumor promotion by these agents is not mediated through alterations in the levels of enzymatic activities which decrease free radical concentrations.« less

Inhibition of 7,12-dimethylbenz[a]anthracene- and urethan-induced lung tumor formation in A/J mice by long-term treatment with dehydroepiandrosterone

Abstract: Long-term treatment with the adrenal steroid dehydro-epiandrosterone reduces weight gain without suppressing appetite and inhibits the occurrence of 7,12-dimethylbenz[a]anthracene- and urethan-induced lung tumors in A/J mice.

Study of 106 organic and inorganic compounds in the Salmonella /microsome test

Abstract: One hundred and six compounds, subdivided into 12 chemical classes (5 polycyclic aromatic hydrocarbons, 7 epoxides and N-oxides, 5 nitro aromatics and heterocycles, 12 aromatic and heterocyclic amines, 2 azo compounds, 17 hydrazine derivatives, 16 miscellaneous aliphatics, 5 miscellaneous heterocycles, 11 miscellaneous organics, 11 hexavalent and 7 trivalent chromium compounds, 8 miscellaneous inorganics), were studied in the Salmonella/microsome test. Fifty-eight of them (54.7%) were found to be mutagenic and 4 additional compounds (3.8%) yielded a positive response following nitrosation in human gastric juice. The results are presented in a tabulated form, providing the following information for each test compound: (a) mutagenic response in 5 S. typhimurium his- strains (TA1535, TA1537, TA1538, TA98, TA100); (b) range of activity for positive compounds or maximum dose tested for negative compounds (in nmol/plate); (c) mutagenic potency (in revertants/nmol compound), varying over a 6.5 x 10(6)-fold range; (d) effect of S-9 mix containing rat (Aroclor)liver S-9 fractions on the mutagenic response (activation, increase, no change or decrease); (e) remarks concerning the influence of other metabolic systems (up to 10 different rat tissue S-9 fractions, mouse S-9 fractions, human S-9 fractions, cell preparations or biological fluids), the interaction between different compounds, the stability and the formation of mutagenic derivatives in human gastric juice and other experimental details. Overlapping of mutagenicity and carcinogenicity data was not evaluated, since the experimental protocol intentionally included a number of non-carcinogenic mutagens and of non-mutagenic carcinogens, with the aim of explaining in some cases the conflicting nature of in vivo and in vitro conclusions.

Mesotheliomas in rats following inoculation with acid-leached chrysotile asbestos and other mineral fibres

Abstract: The carcinogenicity of untreated UICC chrysotile A, of acid (oxalic and hydrochloric)-leached UICC chrysotile A, of crocidolite and of JM 104 glass fibres has been studied by intrapleural injection into rats. This experiment, carried out on 304 animals, demonstrated that when more than 80% of the Mg had been leached from chrysotile fibres by either hydrochloric or oxalic acid, the proportion of pleural mesotheliomas was either nil or dramatically lower than that obtained with untreated chrysotile. The carcinogenic effect of crocidolite was higher than that of 43.7% oxalic acid-leached chrysotile. The proportion of mesotheliomas observed in animals injected with JM 104 glass fibres was similar to that in animals injected with fibres from which 63.8% had been leached with oxalic acid. These results indicate that with regard to the induction of pleural carcinogenicity by chrysotile fibres, not only size characteristics but also parameters such as chemical composition and physiochemical properties must intervene.

Development of hepatocellular carcinomas and increased peroxisomal fatty acid β-oxidation in rats fed [4-chloro-6-(2, 3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14, 643) in the semipurified diet

Abstract: To eliminate interference by contaminating xenobiotics that may possibly be present in the commercial rodent chow, we used semipurified diet in these studied to establish the carcinogenicity of hepatic peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643). This compound was fed to male F344 rats in the semipurified diet at a dietary concentration of 0.2% (w/w) for 65 weeks. Between 40 and 65 weeks, 14 of 14 rats fed Wy-14,643 developed hepatocellular carcinomas. Therefore, the possibility that peroxisome proliferators increase the liver tumor incidence by as promoting effect appears highly unlikely, even though these compounds appear to be non-genotoxic. The liver tumors, as well as non-tumor portions of liver in Wy-14,643 fed rats, showed increased levels of peroxisomal fatty acid beta-oxidation system and H2O2. Excessive accumulation of autofluorescent lipofuscin, indicative of increased lipid peroxidation, was also observed in the liver parenchymal cells, during Wy-14,643 induced liver tumorigenesis. These observations support the contention that sustained proliferation of peroxisomes leads to oxygen radical toxicity, which may eventually lead to the development of liver tumors in rodents exposed to peroxisome proliferators.

Early appearance of histochemically altered hepatocyte foci and liver tumors in female rats treated with carcinogens one day after birth

Abstract: A single i.p. injection of diethylnitrosamine (DEN) or benzo(a)pyrene (BAP) in 1-day-old female rats produced a high incidence of ..gamma..-glutamyltranspeptidase-(GGT)- positive hepatocyte foci within 4 weeks after the rats were weaned onto a 0.05% phenobarbital diet; the injection of benzo(e)pyrene did not produce foci under these conditions. Liver tumors appeared in rats treated with DEN within 8 weeks after weaning and in BAP-treated rats within 16 weeks after weaning. The results suggest that the treatment protocol used in this study may increase the utility of the liver tumorigenesis model as a broadly applicable in vitro system for the rapid detection of tumorigenic potential in environmental contaminants.

5-Methoxypsoralen, the melanogenic additive in sun-tan preparations, is tumorigenic in mice exposed to 365 nm u.v. radiation.

Abstract: 5-Methoxypsoralen (5 MOP), the melanogenic component present in several suntan preparations was synthesized and tested by topical applications in inbred XVII nc/Z albino mice combined with 365 nm irradiation with the aim of establishing the relative carcinogenic activity of this compound, as compared to 8-methoxypsoralen (8 MOP) and psoralen. 85% of the animals developed tumors and 25% had multiple tumors. Additional treatment with 12-O-tetradecanoylphorbol-13-acetate raised the tumor incidence to 100%. Tumors caused by 5 MOP show much longer latent periods than those induced by psoralen and 8 MOP. Most of the tumors were rapidly growing squamous cell carcinomas giving metastases in 20% of the animals. The possible long-term effects that might follow the use of 5 MOP in humans are discussed.

The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures.

Abstract: Primary cultures of rat hepatocytes were exposed to [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) for 4 h, and the RNA and DNA nucleoside adducts were isolated and identified by h.p.l.c. The DNA adducts were shown to be N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG-N2-AAF), while the RNA adducts were N-(guanosin-8-yl)-2-acetyl-aminofluorene, and N-(guanosin-8-yl)-2-aminofluorene. The removal of these adducts was measured up to 38 h following the cessation of exposure of the hepatocytes to N-OH-AAF. The dG-C8-AAF adduct was removed with a half-life of approximately 10 h, while dG-N2-AAF and dG-C8-AF remained constant for 14 h, followed by a slow rate of removal. The dG-C8-AAF adduct initially composed about 60% of the total DNA adducts of primary hepatocytes in contrast to the 20% found in liver in vivo. The formation of the 3 DNA adducts and the different rates of repair indicate that primary cultured rat hepatocytes may be a valuable system to study initiation of liver carcinogenesis by N-OH-AAF.

Selenium-mediated inhibition of 7,12-dimethylbenz[a]anthracene-induced mouse mammary tumorigenesis

Abstract: The effect of supplemental selenium on 7,12-dimethylbenz[a] anthracene (DMBA)-induced mammary tumorigenesis was investigated in several mouse strains. Selenium, administered as SeO2 in the drinking water, inhibited mammary tumor formation in DMBA-treated (C57BL x DBA/2f)F1, C3H/StWi and BALB/c female mice. In addition, selenium inhibited the occurrence of DMBA-induced ductal hyperplasias in (C57BL x DBA/2f)F1 and BALB/c mice and mammary tumour virus-induced alveolar hyperplasias in BALB/cfC3H mice. Selenium did not alter the growth of established mammary tumors. These results demonstrate that supplemental selenium inhibits both chemical-and viral-induced mouse mammary tumorigenesis, and secondly, that the development of preneoplastic lesions, an early stage in mammary tumorigenesis, is very sensitive to selenium-mediated inhibition.

Autoradiography of "oval cells" appearing rapidly in the livers of rats fed N-2-fluorenylacetamide in a choline devoid diet.

Abstract: Autoradiographic analysis of liver sections from rats fed the hepatocarcinogen N-2-fluorenylacetamide (FAA) in a choline devoid (CD) diet suggests that proliferating small "oval" cells arise from a few small portally-situated cells, and spread rapidly across the entire liver lobule Small cells with detectable grains are first located where liver plates meet the portal areas This cell type gradually increases in number over a 10-12 day period, then proliferates rapidly After 28 days, microscopic nodules consisting of heavily labeled large eosinophilic cells appear, whereas residual hepatocytes are not labeled Combined immunofluorescent and autoradiographic labeling studies reveal that many of the small cells contain AFP; approximately half of the alpha-fetoprotein-containing cells are labeled with [3H]thymidine (dT) Feeding CD-FAA diets to rats with hepatocytes prelabeled with [3H]dT after 70% hepatectomy 7 weeks earlier provides data which suggest that small "oval" cells do not arise from prelabeled hepatocytes but, instead, infiltrate the prelabeled hepatocytes during the diet induced proliferative phase We conclude that "oval" cells arise from a small number of portal cells, not from hepatocytes Exact identification of the oval cell precursor is not possible, but it could be a "stem" cell Although hepatocyte-like properties are found in small cells (eg, albumin staining), there is no evidence that they differentiate into normally functioning hepatocytes

DNA-damaging activity in vivo and bacterial mutagenicity of sixteen aromatic amines and azo-derivatives, as related quantitatively to their carcinogenicity.

Abstract: Sixteen aromatic amines and azo-derivatives were studied. They were: benzidine; 2-acetylaminofluorene; 3'-methyl-p-dimethylaminobenzene; o-aminoazo-toluene; p-dimethylaminoazobenzene; 2,4-diamino-toluene; 4,4'-oxydianiline; 2,4-diaminoanisole; 4,4'-methylenedianiline; 2-naphthylamine; Auramine O; Rhodamine B; Ponceau MX; 1-naphthylamine; p-aminoazobenzene and aniline. The compounds were examined for their capability to induce alkaline DNA fragmentation in rat liver after treatment in vivo, for their mutagenicity in the Salmonella strains TA 98 and TA 100, for their acute toxicity and for their carcinogenicity in mice and rats. For each parameter a quantitative potency index was established, and the correlation existing amongst the different parameters investigated. Only mutagenicity in the strain TA 98 was slightly correlated with carcinogenic potency (r = 0.408). DNA fragmentation and toxicity were not correlated with carcinogenicity. A significant correlation was found between DNA fragmentation and toxicity (r = 0.539). No correlation was found between DNA fragmentation and mutagenicity. The lack of correlation between DNA fragmentation and carcinogenicity is in contrast with previous results obtained with a family of hydrazine derivatives (12) and a group of nitrosocompounds (22). For these two groups of chemicals correlation between DNA fragmentation and carcinogenicity existed, but not between carcinogenicity and mutagenicity in the Ames' test. It is suggested that short term tests can perform very differently for different classes of chemicals.

Minor products from the reaction of (+) and (-) benzo[a]-pyrene-anti-diolepoxide with DNA.

Abstract: The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP-diolepoxide) with deoxyguanosine has been studied. In addition to the expected N2-guanine derivative minor products resulting from reaction at the O6 and 7-positions have been identified. Reaction of racemic, (+) or (-) BP-diolepoxide with [14C] and [3H]purine labelled DNA allowed these same products to be identified and their yields estimated. It was found that the O6 and 7-guanine products were derived mainly from reaction of the (-)isomer. The 7-substituted guanine derivative in DNA was unstable, undergoing either spontaneous release of the substituted guanine or imidazole ring opening.

Chemoprevention of mammary tumorigenesis by a combined regimen of selenium and vitamin A

Abstract: Mammary tumorigenesis induced in rats by 7,12-dimethylbenz[a]anthracene was markedly suppressed by combined dietary supplementation with sodium selenite and retinyl acetate; final tumor yield was reduced to 8% of control as compared with 51% and 36%, respectively, for selenium and retinyl acetate alone. A continuous intake of both agents was necessary to sustain the chemopreventive effect.

Anticarcinogenic effect of selenium in rats treated with dimethylbenz[a]anthracene and fed different levels and types of fat.

Abstract: The present investigation reports the effect of selenium supplementation on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis in rats fed either a 5% or a 25% corn oil diet. A reduction in tumorigenesis in both groups was observed with 2.5 p.p.m. of dietary selenium. Selenium supplementation also inhibited the development of hyperplastic alveolar nodules in the mammary gland subsequent to DMBA treatment. In addition, the appearance of mammary neoplasia was reduced by selenium in rats fed a high-saturated fat diet (coconut oil), indicating that the type of fat consumed did not influence the antitumorigenic effectiveness of selenium. The lack of a correlation between the anticarciongenic efficacy of selenium and its ability to suppress lipid peroxidation in the mammary tissue of rats fed either a high-saturated fat or a high-unsaturated fat diet suggests that the inhibitory action of selenium is probably not mediated by its antioxidant function in lipid metabolism

Carcinogen-mediated induction of SV40 DNA synthesis in SV40 transformed Chinese hamster embryo cells

Abstract: Exposure of SV40-transformed Chinese hamster embryo cells (line CO50) to a series of physical and chemical carcinogens (including activation-dependent and activation-independent varieties) resulted in the induction of viral DNA synthesis. The carcinogen mediated amplification of SV40 DNA was demonstrated by a highly sensitive in situ hybridization procedure for the detection of cells synthesizing SV40 DNA. Treatment of CO50 cells with an inhibitor of polycyclic hydrocarbon metabolism (7,8-benzoflavone) prior to the application of benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene prevented the induction of SV40 DNA synthesis, indicating that the induction depends upon the metabolic activation of these compounds. Non-carcinogenic hydrocarbons were inactive under this assay. Two different protocols for determining the inducing potential of a compound are presented. The properties of this test and its possible use as a short-term assay for potential carcinogens is discussed. The possibility that the induction of SV40 DNA synthesis is a reflection of a general gene amplification phenomenon mediated by carcinogens is discussed.

DDT acceleration of mammary gland tumors induced in the male Sprague-Dawley rat by 2-acetamidophenanthrene

Abstract: 2-Acetamidophenanthrene (AAP) yields adducts to rat liver DNA and RNA in amounts comparable to those found for the potent hepatocarcinogen 2-acetamidofluorene, but is not hepatocarcinogenic. This suggested that AAP might initiate liver tumors, but was incapable of causing their progression to a detectable state. To test this hypothesis, the protocol devised by Peraino was used, in which 21-day-old male Sprague-Dawley rats were fed 0.02% AAP in a grain diet for three weeks. this was followed by long-term feeding of 0.05% 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT). The mean latent period of all tumors (primarily mammary tumors) was reduced about six months by the DDT feeding. No tumors were found in rats treated with DDT only. Livers in all animals appeared normal at autopsy or on laparotomy, and showed barely detectable signs of toxicity upon histological examination. Thus, we have found that a once wide-spread environmental chemical acts as a tumor accelerator on a major target for human tumors. Because this finding is in the male rat, the significance of this result for breast cancer in women is uncertain.

A new protocol and its rationale for the study of initiation and promotion of carcinogenesis in rat liver.

Abstract: A new protocol for carcinogenesis in rat liver is described in order that confirmatory experiments might be undertaken concurrently. The basic protocol, designated IPI (initiator + promoter + initiator), is presented in several alternative forms including the possible use of X-irradiation as the initiator. The rationale is discussed in terms of the two-hit somatic mutation theory of Armitage and Doll, with an initial hit produced by the first dose of initiator and expansion of single cells to sizable clones by promotion thereby increasing the probability of a second hit by the second dose of initiator. The question of relevant mutations is taken up and it is proposed that genes for chalones and for chalone receptors are logical targets for consideration in a two-mutation sequence.

Rat liver microsomal azoreductase activity on four azo dyes derived from benzidine, 3,3'-dimethylbenzidine or 3,3'-dimethoxybenzidine.

Abstract: The ability of rat liver microsomes from phenobarbitone pretreated animals to reduce the azo groups of amaranth, sunset yellow, congo red, trypan blue, chloramine sky blue FF and direct black 38 was measured spectrophotometrically in vitro. The dyes amaranth and sunset yellow acted as positive controls. Of the dyes derived from benzidine or its congeners, only direct black 38 was reduced to an appreciable extent; the rate of reduction was 10% of that for amaranth. The dyes were tested for mutagenicity in the Salmonella/microsome assay, the only active compound being direct black 38. Mutagenicity of this dye may be due in part to the mutagen 1,2,4-triaminobenzene. Mutagenic activity and azo-reduction of direct black 38 was independent of the presence of oxygen. The results presented suggest that mammalian liver may play only a minor or negligible role in the azo-reduction of dyes derived from benzidine or its congeners.

Metabolism of 1-nitropyrene and formation of DNA adducts in Salmonella typhimurium

Abstract: 1-Nitropyrene is slowly reduced by intact cells of Salmonella typhimurium to yield 1-aminopyrene and N-acetyl-1-aminopyrene plus six unidentified minor products. When the bacteria are exposed to tritiated 1-nitropyrene, increasing amounts of radioactivity become bound to DNA as the nitropyrene is metabolized. Enzymatic hydrolysis of the labelled DNA yields low molecular weight labelled compounds which probably represent nucleoside adducts formed by the reaction of nitropyrene metabolites with DNA. Results with appropriate mutant strains indicate that bacterial nitroreductases are involved in activating nitropyrene to a reactive intermediate that binds to DNA and that nitropyrene adducts in DNA are subject to excision repair.

Assessment of the carcinogenic N-nitrosodiethanolamine in tobacco products and tobacco smoke

Abstract: A simple, reproducible gas chromatography-thermal energy analyzer (g.c.-TEA) method has been developed for the analysis of N-nitrosodiethanolamine (NDELA) in tobacco and tobacco smoke. The extract of tobacco or the trapped particulates of tobacco smoke are chromatographed on silica gel. The NDELA containing fractions are concentrated, silylated and analyzed with a modified g.c.-TEA system. (/sup 14/C)NDELA serves as internal standard for the quantitative analysis. Experimental cigarettes made from tobaccos which were treated with the sucker growth inhibitor maleic hydrazidediethanolamine (MH-DELA) contained 115--420 p.p.b. of NDELA and their smoke contained 20--290 ng/cigarette, whereas hand-suckered tobacco and its smoke were free of NDELA. The tobacco of US smoking products contained 115--420 p.p.b. of NDELA and the mainstream smoke from such products yielded 10--68 ng/cigar or cigarette. NDELA levels in chewing tobacco ranged from 220--280 p.p.b. and in two commercial snuff products were 3,200 and 6,800 p.p.b. Although the five analyzed MH-DELA preparations contained between 0.6--1.9 p.p.m. NDELA it is evident that the major portion of NDELA in tobacco is formed from the DELA residue during the tobacco processing. Based on bioassay data from various laboratories which have shown that NDELA is a relatively strong carcinogen and based on the results of this studymore » the use of MH-DELA for the cultivation of tobacco is questioned.« less

Tissue-dependent enzyme-mediated repair or removal of O-ethyl pyrimidines and ethyl purines in carcinogen-treated rats

Abstract: Treatment of perinatal rats with N-ethyl-N-nitrosourea (EtNU) leads predominantly to brain tumors. The DNA in tissues of 10-day-old BD IX rats is alkylated by this ultimate carcinogen at the same sites as is DNA in mammalian cell cultures or DNA in solution. Similar proportions of the derivatives quantitated (O6-EtG, 7-EtG, 3-EtA, O2-EtT, O4EtT, O2EtC, and ethyl phosphotriesters) are found in each tissue examined 1 h after treatment with EtNU. Most of the ethylated bases are poorly removed (or, in the case of O4-EtT, not at all) from DNA in the brain, the target tissue of oncogenicity. A pool of five other tissues, excluding liver, exhibits a similar pattern of ethyl base persistence over a 75 h period. In contrast, liver apparently contains enzymes capable of removing all of the ethylated bases. In all tissues used, ethyl phosphotriesters are very stable. The observed kinetics imply that removal of ethylated bases would be complete within 10 days in liver, while over 50% of the chemically ethylated stable bases would persist in other tissues, including brain, for many weeks. We propose that any or all persistent promutagenic derivatives (O6-EtG, O2EtT, O4-EtT, O2-EtC) can be important in the initiation of carcinogenesis by somatic mutation, given that the damage DNA is expressed. The differing rates of removal of the ethyl purines and pyrimidines in brain, liver and pooled tissues imply that mammals possess multiple independent repair systems.

Role of sulfation in the formation of DNA adducts from N-hydroxy-2-acetylaminofluorene in rat liver in vivo . Inhibition of N-acetylated aminofluorene adduct formation by penta-chlorophenol

Abstract: N-Hydroxy-2-acetylaminofluorene (N-OH-AAf) is metabolically converted into reactive N,O-esters which are capable of forming covalent adducts with DNA in rat liver in vivo. The effect of inhibiting one of the proposed pathways, N-O-sulfation, on DNA adduct formation was studied by using a specific sulfotransferase inhibitor, pentachlorophenol. Rats were pretreated with pentachlorophenol and, after 45 min, N-OH-AAF was administered. Four hours after dosing the animals were sacrificed and hepatic DNA was isolated. In DNA from control livers two acetylaminofluorene-and one aminofluorene-substituted deoxyguanosine adducts were found. The acetylaminofluorene derivatives, N-(deoxyguanosin-8-yl)-2--acetylaminofluorene and 3-(deoxyguanosin-N2-yl)-2acetylaminofluorene, accounted for 40% of the total binding in the hydrolyzed DNA. The aminofluorene adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene, accounted for the remainder. In rats that were pretreated with pentacholorphenol, total DNA binding was decreased by 26%. The same three adducts were found, but the acetylaminofluorene adducts were now only 13% of the total, while the aminofluorene adduct accounted for 87%. The absolute amount of aminofluorene adduct was not altered as compared to control rats. These data demonstrate the involvement of N-O-sulfation in carcinogen-DNA binding and indicate that at least 70% of the acetylaminofluorene bound to deoxyguanosine in rat liver DNA, in vivo, is formed through N-O-sulfation of N-OH-AAF.

Characterization of the hepatic DNA damage caused by 1,2-dibromoethane using the alkaline elution technique.

Abstract: The damage to hepatic cell DNA caused by i.p. administration of 1,2-dibromoethane (EDB) was studied in male Swiss Webster mice. Three hours after treatment, hepatic nuclei were isolated and damage to DNA assessed by the alkaline elution technique. The method for isolation of the nuclei preserved the integrity of the DNA and in addition it was found that the purified nuclei could be frozen for at least 1 week with no detectable damage to the DNA. EDB administration (25-75 mg/kg) resulted in a dose-dependent increase in DNA single-strand breaks. More DNA single-strand breaks were detected when lysed nuclei were preincubated in the alkaline eluting solution prior to analysis. The presence of these alkali-labile sites suggests that the DNA strand breaks result, in part, from the lability of DNA sites alkylated by EDB. There was no evidence of EDB induced DNA-DNA cross-links or DNA-protein cross-links. The use of isolated hepatic nuclei as a sample for alkaline elution analysis may be a useful technique for studying the nature of DNA damage induced in vivo by carcinogens.

Hypomethylation of DNA in Raji cells after treatment with N-methyl-N-nitrosourea.

Abstract: Human Raji lymphoblast-like cells were propagated in the presence of various concentrations of N-methyl-N-nitrosourea (MNU) and the degree of enzymatic methylation of newly synthesized DNA was analysed by two independent methods. The overall extent of enzymatic DNA methylation was measured on the basis of [14C]deoxycytidine derived radioactivity incorporated into DNA 5-methylcytosine and cytosine residues. Enzymatic methylation of internal cytosines at 5'-CCGG-3' sequences of Raji DNA was analysed by use of the bacterial restriction enzyme HpaII and its isoschizomer MspI. The data obtained by both methods indicate that the treatment with MNU causes a lower level of enzymatic methylation of newly synthesized DNA. This lower extent of DNA methylation persists in the absence of the carcinogen in the cell cycles following the treatment.

DNA methylation and methylase levels in normal and malignant mouse hepatic tissues

Abstract: The status of DNA methylation, as measured by the 5-methylcytosine content of nuclear DNA, was examined in normal livers and in chemically induced or spontaneous primary hepatocellular carcinoma (PHC) arising in three strains of mice. The DNA from spontaneous tumors of genetic origin in C3H mice and also from acetylaminofluorene, chlordane, or 3'-methyl-4-dimethylaminoazobenzene-induced tumors in C57Bl and B6C3 mice was undermethylated compared to the levels in background and normal liver samples. The DNA methylase activities from normal liver, background liver, and PHC were assayed in C3H mice to determine whether the observed genomic undermethylation is related to a dysfunction of this enzyme and were compared to the rates of DNA synthesis in these tissues. Since DNA methylase levels from tumor nuclei were elevated compared to background, it is concluded that the undermethylation found in the tumor genomes of this system is not due to inactivation nor a significant deficiency of the activity of this enzyme relative to the demand in tumors for methylation of de novo synthesized DNA.