Showing papers in "Carcinogenesis in 1980"
TL;DR: The methods described provide reproducible and quantitative methods of analysis for all the known methylated or ethylated products in a single DNA sample.
Abstract: Methods were developed for the efficient routine degradation and fractionation of ethylated and methylated DNA. Alkylated DNA was hydrolyzed by a neutral thermal method to yield 3- and 7- alkylpurines and O2-alkylcytosines. The partially apurinic DNA was separated from the bases by precipitation in 0.1 N HCl. Portions of the DNA precipitate were further hydrolyzed either by 0.1 N HCl to yield purine bases, or by enzymes to yield nucleosides and phosphotriesters. The chemical and enzymic digests were fractionated by a combination of high pressure liquid chromatography systems to yield quantitative estimates of the following products from methylated or ethylated DNA: 1-, 3-, and 7-alkyladenines, O2-alkylcytosines, 3-, O6-, and 7- alkylguanines and O2-, 3-, and O4-alkylthymines. N6-Alkyladenines, 1-alkylguanines and N2-alkylguanines were not detected and the 3- alkylcytosines were detected but not quantified. Phosphotriesters were estimated from the amounts of recovered alkyl phosphotriesters of thymidylyl (3'-5') thymidine. Using these methods, it was possible to account for 98, 81, 98, and 92% of the DNA bound alkyl groups obtained from DNA reacted with [14C]methyl methanesulfonate, [3H]ethyl methanesulfonate, N-[3H]-methyl-N-nitrosourea, and N-[14C]ethyl-N-nitrosourea, respectively. The methods described provide reproducible and quantitative methods of analysis for all the known methylated or ethylated products in a single DNA sample.
288 citations
TL;DR: Two human astrocytoma cell strains as defective in the repair of N-methyl-N' -nitro-N-nitrosoguanidine (MNNG) damaged adenovirus 5 are shown to be very sensitive to MNNG-produced killing as measured by colony forming ability, but are normally sensitive to ultraviolet light.
Abstract: We have previously identified four human astrocytoma cell strains as defective in the repair of N-methyl-N' -nitro-N-nitrosoguanidine (MNNG) damaged adenovirus 5. We now show that two of these strains (the only two tested), in comparison to other tumor strains or normal human skin fibroblasts, are very sensitive to MNNG-produced killing as measured by colony forming ability, but are normally sensitive to ultraviolet light. Further, such repair deficient cells may be cultured from tumors of the colon, lung, skin, and neck. The phenotype of deficient repair of MNNG-treated adenovirus 5 has now been found in a subgroup of 9 of the 39 human tumor strains tested. We propose to call this phenotype the Mer(-) phenotype. None of the 22 strains of normal human skin fibroblasts tested showed deficient repair of MNNG damage. MNNG treatment (80 microM) causes a decrease in semi-conservative DNA synthesis from which Mer(-) tumor cells do not recover, but from which cells capable of normal repair of MNNG damage (Mer(+)) do. Somewhat paradoxically, Mer(-) cells show more MNNG-stimulated DNA synthesis ('repair synthesis') than do Mer(+) cells. Besides being deficient in the repair of MNNG-damaged adenoviruses Mer(-) cells also have difficulty in repairing viruses damaged either by other N-alkyl-N'-nitro-N-nitrosoguanidines, or by N-methyl- or N-ethyl-N-nitrosoureas.
164 citations
TL;DR: Results indicate that this assay is a potentially useful system for assessing the genotoxic and potential carcinogenic activity of chemicals in the whole animal and a linear decline in UDS during the first 24 h post-treatment followed by a slower decline from 24 to 48 h.
Abstract: An assay is described for the measurement of chemically-induced DNA repair in cultures of primary rat hepatocytes following in vivo treatment with genotoxic agents. Rats were exposed to chemicals then primary hepatocytes were isolated by liver perfusion and cultured with [3H]-thymidine. DNA repair was measured as unscheduled DNA synthesis (UDS) by quantitative autoradiography. Cells from control animals consistently ranged from -2 to -6 net grains (NG). Treatment of rats with 10, 1 or 0.1 mg/kg dimethylnitrosamine (DMN), i.p., yielded 36.6, 6.4 and -0.9 NG, respectively; 10 mg/kg DMN, per os (p.o.), produced 22.2 NG. Oral doses of 50 or 5 mg/kg acetylaminofluorene (AAF) yielded 14.0 and 6.4 NG, respectively. Carbon tetrachloride (CCl4) at 100 or 10 mg/kg, p.o., yielded -3.2 and -5.1 NG, respectively. Thus, dose-related increases in UDS were observed for the hepatocarcinogens DMN and AAF while vehicle controls and the hepatotoxin CCl4 produced no response. An examination of the time-course of DNA repair following DMN treatment shows a linear decline in UDS during the first 24 h post-treatment followed by a slower decline from 24 to 48 h. These results indicate that this assay is a potentially useful system for assessing the genotoxic and potential carcinogenic activity of chemicals in the whole animal.
143 citations
TL;DR: Dietaries rich in protease inhibitors may contribute to reducing cancer incidence in man by reducing the induction of mammary cancer in x-irradiated rats.
Abstract: This paper examines the relationship between feeding a diet rich in protease inhibitors and the reduction of mammary cancer induced by x-irradiation in Sprague-Dawley rats. Of a total of 145 irradiated animals, 44% of the 45 rats fed a raw soybean diet containing a high concentration of protease inhibitor developed mammary tumors as compared to 74% of 50 rats fed a casein diet containing no protease inhibitor. Animals fed Purina rat chow which contained low levels of protease inhibitor exhibited a 70% mammary tumor incidence. No spontaneous neoplasms were found in any of the non-irradiated animals on the raw soybean diet whereas about 10% of the animals on the protease-free diet developed tumors. Thus, soybeans which are rich in protease inhibitors reduced the induction of mammary cancer in x-irradiated rats. This work suggests that diets rich in protease inhibitors may contribute to reducing cancer incidence in man.
139 citations
TL;DR: The results indicate that the incidence of papillomas serves as a rapid (18 weeks) index for subsequent appearance of carcinomas, and twice weekly applications of 10 nmol of TPA for 18 weeks following initiation of female CD-1 mice with 0.2 micromol of DMBA is an appropriate protocol for maximum tumor yield in initiation-promotion experiments.
Abstract: The effects of dose and duration of treatment with the potent tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) on the formation of skin tumors in Charles River CD-1 mice was studied. Mice were initiated with a single application of 0.2 micromol of 7,12-dimethylbenz[a]anthracene (DMBA) in 0.2 ml acetone. Beginning two weeks after initiation, mice were treated twice weekly with various doses (0.01 - 20 nmol) of TPA in 0.2 ml acetone. Application of either 0.01 or 0.1 nmol of TPA did not elicit tumors during the 50 weeks duration of treatment. A dose-dependent increase in the number of papillomas was observed through the range of 1 to 10 nmol of TPA. Twice weekly applications of 20 nmol of TPA did not further enhance the papilloma incidence. A good correlation was observed between the induction of ornithine decarboxylase (ODC) activity and the formation of skin tumors by various doses of TPA. To determine the effect of promotion duration on the incidence of papillomas and carcinomas, mice were treated with 10 nmol of TPA for various durations (6, 12, 18, 24, 30, or 36 weeks) beginning 2 weeks after initiation with 0.2 micromol of DMBA. Mice promoted for only 6 weeks developed papillomas and carcinomas after promotion had been discontinued. There was an intermediate incidence of tumors in the group treated for 12 weeks. Promotion for 18, 24, 30, or 36 weeks elicited virtually identical yields of papillomas. The incidence of carcinomas was proportional to promotion duration times of 6, 12, and 18 weeks, but carcinoma incidence was less than maximal in mice promoted for 24 weeks or longer. The results indicate that a) the incidence of papillomas serves as a rapid (18 weeks) index for subsequent appearance of carcinomas, b) twice weekly applications of 10 nmol of TPA for 18 weeks following initiation of female CD-1 mice with 0.2 micromol of DMBA is an appropriate protocol for maximum tumor yield in initiation-promotion experiments, and c) ODC induction may be an important component of the mechanism of skin tumor promotion by TPA.
119 citations
115 citations
TL;DR: This cell line will be useful for studying metabolic activation of polycyclic aromatic hydrocarbons and other xenobiotics by human tissue and as an activation system in short-term screening assays for identifying compounds with carcinogenic potential for humans.
Abstract: The liver-derived human cell line, Hep G2, has high benzo[a]pyrene-metabolizing activity and converts benzo[a]pyrene to intermediates that are mutagenic and that bind to DNA. This cell line will be useful for studying metabolic activation of polycyclic aromatic hydrocarbons and other xenobiotics by human tissue and as an activation system in short-term screening assays for identifying compounds with carcinogenic potential for humans.
104 citations
TL;DR: The probable ultimate urinary bladder carcinogen, N-hydroxy- 2-naphthylamine (N-HO-2-NA), reacted with nucleic acids and proteins under mildly acidic conditions (pH 5) to form covalently bound derivatives, and their possible role in the initiation of carcinogenesis is discussed.
Abstract: The probable ultimate urinary bladder carcinogen, N-hydroxy-2-naphthylamine (N-HO-2-NA), reacted with nucleic acids and proteins under mildly acidic conditions (pH 5) to form covalently bound derivatives. The extent of reaction was in the order: polyguanylic acid > DNA > or = protein > rRNA > tRNA > polyadenylic acid, polyuridylic acid > polycytidylic acid. At pH 7, appreciable reaction occurred only with protein. Enzymatic hydrolyses of the DNA, which contained 1.5 naphthyl residues/1000 nucleotides, yielded 3 nucleoside-arylamine adducts. From chemical, u.v., n.m.r., and mass spectrometric analyses, the adducts were identified as 1-(deoxyguanosin-N2-yl)-2-naphthylamine, 1-(deoxyadenosin-N6-yl)-2-naphthylamine, and a purine ring-opened derivative of N-(deoxyguanosin-8-yl)-2-naphthylamine, tentatively identified as 1-[5-(2,6-diamino-4-oxopyrimidinyl-N6-deoxyriboside)]-3-(2-naphthyl)urea. The properties of these adducts and their possible role in the initiation of carcinogenesis are discussed.
90 citations
TL;DR: This model system represents a quantitative assay for carcinogen altered epithelial cell differentiation and may select for an early property of preneoplastic epidermal cells.
Abstract: Basal epidermal cells can be selectively maintained as a monolayer in culture medium containing a low ionic calcium concentration of 0.01-0.10 mM. Cessation of proliferation, maturation and shedding of squamous sheets can be induced in this population by increasing the calcium concentration above 0.1 mM. Since alterations in the regulation of proliferation and differentiation are associated with epidermal carcinogenesis in vivo, it appeared reasonable that changes in the phenotypic response to calcium might follow exposure to carcinogens in vitro. Support for this hypothesis was provided by the observation that malignant epidermal cells continued to proliferate when switched from low to high calcium medium, and could thus be selected from a mixture of such cells and a large excess of normal cells which did not survive after induced differentiation. Normal primary epidermal cells were plated in low calcium medium, treated on day 3 with a chemical carcinogen, maintained for 3-9 weeks in low calcium (0.02 mM) and then switched to high calcium medium (1.4 mM). After an additional 4 weeks, surviving epithelial colonies were fixed, stained with rhodamine and counted. Treatment of cultures with 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine yielded 4-10 fold more colonies than solvent controls. Colony number was proportional to carcinogen dose for both agents, and increased with time in low calcium prior to selection by calcium increase. Cells obtained from colonies in treated cultures demonstrated characteristic epidermal morphology and keratinization, and could be subcultured, but did not grow in agar or produce tumors in syngeneic hosts. This model system represents a quantitative assay for carcinogen altered epithelial cell differentiation and may select for an early property of preneoplastic epidermal cells.
89 citations
TL;DR: Two-dimensional gels of the keratin polypeptides of normal epidermis and of benign tumors exhibited spot patterns which could be divided at the 61,000 dalton level into an essentially basic subgroup, comprising the high molecular weight keratins, and an acidic subgroup including the low molecular weight components.
Abstract: A comparative study of the keratin composition of adult and neonatal mouse epidermis, benign and malignant tumors of mouse skin and murine epidermal cells grown in culture revealed striking differences in the keratin polypeptide patterns when analyzed by one-dimensional gel electrophoresis. Not only did the study confirm body-site specific alterations in the keratin patterns within one species, but it also demonstrated that similar to cultured epidermal cells, three malignant skin tumors investigated specifically lacked a group of keratin components with molecular weights larger than 61,000 daltons, which were invariably present in all normal and also in benign tissues. These findings offer the possibility of using keratins as molecular markers of the malignant state of epidermal cells. Two-dimensional gels of the keratin polypeptides of normal epidermis and of benign tumors exhibited spot patterns which could be divided at the 61,000 dalton level into an essentially basic subgroup, comprising the high molecular weight keratins, and an acidic subgroup including the low molecular weight components. The two uppermost proteins of cultured epidermal cells and carcinomas (molecular weights 61,000 and 58,000 daltons) belong to the acidic subgroup in normal tissues as well as in papillomas. However, in the case of malignant tumors and in vitro transformed epidermal cells they showed distinct alterations in charge in that they migrated into the more alkaline part of the gel. The number of spots appearing in the more basic region of the gel could be inversely related to the degree of differentiation exhibited by tumors or in vitro cells.
87 citations
TL;DR: Different dark-cell inducing characteristics seem to be the only detectable difference in early effects produced by TPA and MZ and would point to the importance of the production of the dedifferentiated dark cells during early stages of tumor promotion.
Abstract: 12-O-Tetradecanoylphorbol-13-acetate (TPA) and mezerein (MZ) are diterpene esters of similar structure and approximately equipotent on a molar basis as far as their hyperplasiogenic, inflammatogenic, and induction of ornithine decarboxylase activity effects in mouse skin are concerned. On the other hand, TPA is much more effective than MZ as a tumor promoter. The percentage of dark basal keratinocytes was determined in the interfollicular epidermis (IFE) of mice topically treated with 1, 2, or 4 ..mu..g of either TPA or MZ in a single application and studied at 12, 24, 48, 96, and 144 h thereafter. The results showed that TPA induced 2 to 3 times more dark cells than MZ in the IFE as well as in the infundibular portion of the hair follicle. The latter epithelium presented a larger number of dark keratinocytes than the IFE in all experimental and control situations, and the differences between the effects of TPA and MZ were even greater in the infundibular epidermis than in the IFE. TPA induced an increase of 5 to 11 times over the control number of dark cells (approx. 2% in IFE), reaching maximum values of 21% in the basal layer 24 h after topical application of 4 ..mu..gmore » of TPA. MZ only produced a 3- to 6-fold increment. The labeling indices of the basal layer and of the dark basal cells were markedly and similarly increased with both compounds. The different dark-cell inducing characteristics seem to be the only detectable difference in early effects produced by TPA and MZ and would point to the importance of the production of the dedifferentiated dark cells during early stages of tumor promotion.« less
TL;DR: A possible structure of modified DNA was proposed which had less perturbation of the helix than that of the acetylated adduct, consistent with single strand endonuclease hydrolysis data.
Abstract: The conformation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene and N-(deoxyguanosin-8-yl)-2-aminofluorene was investigated by 1H n.m.r. spectroscopy. There was rotation about the glycosyl bond and preference for either the anti or syn conformation depended on whether or not the 8-arylamino nitrogen was acetylated. The unacetylated adduct existed preferentially in the anti form and the acetylated adduct existed preferentially in the alternate syn form. There was also rotation about the backbone C4' -C5' bond. The unacetylated adduct was mainly in the gauche-gauche conformation about C4'-C5', while the acetylated adduct was mainly in the alternate gauche-trans/trans-gauche form. Using space filling models with the conformation of the unacetylated adduct conserved in the helix, a possible structure of modified DNA was proposed which had less perturbation of the helix than that of the acetylated adduct. This was consistent with single strand endonuclease hydrolysis data. The acetylated and unacetylated adducts may cause entirely different types of local conformational changes in DNA because of major differences in interactions between the base and the sugar moiety at the modified nucleoside level.
TL;DR: It is demonstrated that dietary selenium prevents and retards tumor development only as long as it is supplied in adequate amounts, consistent with its role as a non-accumulative trace nutrient.
Abstract: Female inbred C3H/St mice infected with the Bittner Milk Particle, develop mammary adenocarcinoma with 27% incidence if maintained on a Torula Yeast diet supplemented with 1 ppm of selenium (organically bound, in yeast). Animals switched from the 1 ppm Se diet to a diet containing only 0.15 ppm Se after reaching the age of 13.8 months develop mammary tumors rapidly during their remaining lifespan, the overall tumor incidence reaches 69%, not statistically different from the 77% incidence of tumors observed in animals maintained on the 0.15 ppm Se diet over their entire post-weaning life span. Conversely, animals changed from the 0.15 ppm Se diet to that containing 1.0 ppm Se at the age of 13.8 months develop mammary tumors with a total incidence of only 46%, significantly lower (P < 0.05) than in the 0.15 ppm Se control group. This study demonstrates that dietary selenium prevents and retards tumor development only as long as it is supplied in adequate amounts, consistent with its role as a non-accumulative trace nutrient.
TL;DR: XP3BR cells were more sensitive than normal cells to the lethal action not only of u.v. but also of gamma irradiation, in contrast to all other XP cells tested to date including XP2BI, the other representative of complementation group G.
Abstract: XP3BR is a fibroblast strain derived from a xeroderma pigmentosum patient exhibiting severe mental retardation in addition to the typical changes in the skin. No tumours have been observed by 6 years of age. Cells from this patient had no detectable excision repair of u.v. damage. The defect in daughter strand repair was also characteristic of excision-defective XP's. The material was assigned to complementation group G and is the second (unrelated) example from this group. XP3BR cells were more sensitive than normal cells to the lethal action not only of u.v. but also of gamma irradiation, in contrast to all other XP cells tested to date including XP2BI, the other representative of complementation group G. The u.v. sensitivity was similar to that of strains from complementation groups A and D, confirming the correlation between extreme u.v. sensitivity and the presence of neurological defects. Following treatment with u.v., XP3BR, and other XPs gave more 6-thioguanine resistant mutants than normal cells whether the comparison was made per unit of dose or per lethal event. After low doses of gamma irradiation XP3BR cells were more mutable than normal or XP2BI cells.
TL;DR: Kinetic studies of the hydrolysis reaction showed that it occurs already at a measurable rate at pH 9.5 and 37 degrees C, and the pyrimidine derivatives have been identified as 1-[6-(2,5-diamino-4-oxopyrimidinyl-N6-deoxyriboside)]-3-(2-fluorenyl)ureas, which probably are stereoisomers.
Abstract: The major aminofluorene-DNA derivative formed from the carcinogen N-acetyl-2-aminofluorene in vivo in rat liver is N-(deoxyguanosin-8-yl)-2-aminofluorene. This nucleoside is hydrolyzed in aqueous solution at alkaline pH through the 7-8 guanine bond to form two pyrimidine derivatives which were separated by Sephadex LH-20 column chromatography and thin-layer chromatography on silica. From chemical, u.v., i.r., n.m.r. and mass spectral analysis the pyrimidine derivatives have been identified as 1-[6-(2,5-diamino-4-oxopyrimidinyl-N6-deoxyriboside)]-3-(2-fluorenyl)ureas, which probably are stereoisomers. Similar products were isolated from enzymatic hydrolysates of DNA reacted with N-hydroxy-2-aminofluorene under mildly acidic conditions (pH 5) and subsequent treatment with 0.1 N NaOH. Kinetic studies of the hydrolysis reaction showed that it occurs already at a measurable rate at pH 9.5 and 37 degrees C. The reaction is catalyzed by Mg2+ and Mn2+ ions and by alkaline phosphatase from E. coli.
TL;DR: The data suggest that in the rat the selective induction of oesophageal tumours by N-nitrosomethylbenzylamine and related asymmetrical nitrosamines is mediated by a preferential bioactivation of the carcinogen in the target organ.
Abstract: Male Wistar rats received a single i.v. injection of the oesophageal carcinogen N-nitroso[methyl-14C]-methylbenzylnitrosamine (2.5 mg/kg body weight). Rapid distribution of the carcinogen occurred, with highest initial concentrations in liver and kidney. Within 10 min after the injection, 14C-labelled metabolites accounted for 50% of the total radioactivity present in the oesophagus, for approximately 25% in liver and forestomach, and for less than 20% in all other organs investigated. Decay of the carcinogen in rat serum followed first-order kinetics with a half-life of 35 min. Of the total radioactivity administered, 49% was exhaled as 14CO2 within 10 h and an additional 5-10% was excreted via urine and faeces. Four hours after a single i.v. injection of N-nitroso-[methyl-14C]benzylnitrosamine methylation of purine bases in DNA was most extensive in the oesophagus, followed by liver, lung and forestomach DNA. In the remaining tissues, DNA methylation was either considerably less (kidney, glandular stomach, spleen) or not at all detectable (ileum, colon, brain). At this time the concentration of the promutagenic base O6-methylguanine in oesophageal DNA was six times higher than in lung and nine times higher than in hepatic DNA. These data suggest that in the rat the selective induction of oesophageal tumours by N-nitrosomethylbenzylamine and related asymmetrical nitrosamines is mediated by a preferential bioactivation of the carcinogen in the target organ.
TL;DR: Both styrene and its presumed active metabolite styrene oxide show dose response as potent inducers of sister chromatid exchanges (SCEs) in human lymphocyte cultures.
Abstract: Both styrene and its presumed active metabolite styrene oxide show dose response as potent inducers of sister chromatid exchanges (SCEs) in human lymphocyte cultures. The SCE inducing and clastogenic capacity of styrene in lymphocytes in vitro can be explained by gas chromatographically measurable increase of styrene oxide in styrene treated cultures.
TL;DR: Findings of alterations in three membrane-associated enzymes indicate that phenobarbital produces substantial changes in the composition of cellular membranes which may be related to its promoting activity.
Abstract: Administration of phenobarbital for 5 to 7.5 weeks to aged C3HfB/HeN mice with spontaneous liver tumors produced an enhancement of gamma glutamyl transpeptidase activity in the tumors and a decrease in glucose-6-phosphatase and adenosine triphosphatase activity. Discontinuation of phenobarbital feeding for 5.5 weeks resulted in the loss of gamma glutamyl transpeptidase activity in tumors. These findings of alterations in three membrane-associated enzymes indicate that phenobarbital produces substantial changes in the composition of cellular membranes which may be related to its promoting activity.
TL;DR: The results suggest that a similar mechanism for the induction of malignant transformation may be involved for high doses of radiation alone or for a low dose of radiation followed by TPA treatment.
Abstract: We have performed experiments designed to investigate the mechanism for the enhancement of radiation transformation in vitro by 12-O-tetradecanoylphorbol-13-acetate (TPA). Two types of experiments, involving C3H 10T1/2 cells and 100 rad X-ray exposures with subsequent TPA treatment, are reported here. In one set of experiments, cultures were initially seeded at differing initial cell densities prior to irradiation. In the other series of experiments, cultures exposed to 100 rads and TPA contained the same initial cell densities (about 300 viable cells per dish); these cultures were allowed to reach confluence and were then reseeded at various cell densities to allow a second cycle of growth to confluence (which involved different numbers of cell divisions). We have observed that the number of transformed foci which ultimately developed per dish treated with 100 rads and TPA remains approximately constant even though the cell density (initial or reseeded) is varied over several orders of magnitude. The yield of transformants per dish which occurred following this treatment was similar to those previously observed in cultures irradiated with higher doses (400-600 rads) of X-rays alone. Our results suggest that a similar mechanism for the induction of malignant transformation may be involved for high doses of radiation alone or for a low dose of radiation followed by TPA treatment.
TL;DR: What interindividual differences exist in these enzyme activities and whether there is a correlation between the activities of these epoxide forming and metabolizing enzymes in preparations from peripheral lung samples and the occurrence of bronchogenic carcinomas in smokers and non-smokers are studied.
Abstract: Activities of microsomal monooxygenases (MO) and epoxide hydrolase (EH) and cytoplasmic glutathione-S-transferases (GST) will contribute to controlling the pool of reactive intermediates, enzymatically derived from polynuclear aromatic hydrocarbons (PAH) within the cells of target organs such as the human lung. Therefore, we studied what interindividual differences exist in these enzyme activities and whether there is a correlation between the activities of these epoxide forming and metabolizing enzymes in preparations from peripheral lung samples and the occurrence of bronchogenic carcinomas in smokers and non-smokers. 57 samples obtained from surgery were studied. Among them were 12 samples from non-smoking patients without cancer as a control group. It is not known whether this control group behaves, with respect to the investigated parameters, identically to fully healthy people, since in all cases indications existed which justified the removal of lung biopsies. Using very sensitive standard assays with benzo[a]pyrene, biphenyl, 7-ethoxyresorufin and 7-ethoxycoumarin as substrates, MO activity could only be determined as O-deethylation of 7-ethoxycoumarin and only after modification of the assay method. Evidence was obtained for the presence of a diffusible, but not dialysible, MO inhibitor in human lung microsomes. The MO activity (substrate: 7-ethoxycoumarin) in this fraction was extremely low in human (100-fold lower than in rat lung preparations), whereas EH (substrate: benzo[a]pyrene 4,5-oxide) was slightly (about 2-fold) higher in human and GST (substrate: 2,4-dinitrochlorobenzene) had similar activities in both species. Interindividual variations of enzyme activities in human lung were considerable: MO, 40-fold: EH, 5-fold; GST 10-fold. Compared to the control group (non-smokers without cancer) MO activities were slightly but significantly higher in lungs from bronchogenic carcinoma patients whether they were smokers (170% of controls, p 0.1) between the various groups studied. The substrate specificity of human lung EH, which was studied using five K-region epoxides of various PAH as substrates, corresponded to that in human and rat liver and in human, mouse and rat skin and to the pure enzyme isolated from rat liver. In contrast to rat liver hepatoma preparations, where EH had been shown to be increased in the tumor tissue and had been identified as a preneoplastic antigen, EH activity in lung microsomal preparations from samples of peripheral squamous cell carcinomas of two subjects had in the tumor tissue only one third of the activity of non-diseased areas of the same lung.
TL;DR: It was found that the removal of O6-methylguanine did not depend upon xth gene function or the uvr endonuclease, however, the rate of elimination of this product was markedly decreased in polA strains, and the results indicate that the elimination of O 6-methylGuanine, but not of 3-methyladenine, requires protein synthesis.
Abstract: Cultures of Escherichia coli were treated with alkylnitrosoureas. The rates of removal of methylation and ethylation products from the DNA of strains defective in various repair pathways were compared with those of their respective wild-type strains. It was found that the removal of O6-methylguanine did not depend upon xth gene function or the uvr endonuclease. However, the rate of elimination of this product was markedly decreased in polA strains. O6-Ethylguanine (in contrast to its methyl analogue) was removed more slowly from the DNA of uvrA(-) than from that of uvrA(+) strains, indicating that the removal of O6-ethylguanine can be initiated by the uvr endonuclease. The composition of the medium in which methylated cells were resuspended following treatment with N-methyl-N-nitrosourea was also found to influence the rate at which O6-methylguanine was removed from the DNA of treated bacteria. No significant removal of this product from bacterial DNA occurred during treatment of cells in buffer, or when treated bacteria were resuspended in salts medium or in growth medium containing chloramphenicol. The results indicate that the elimination of O6-methylguanine, but not of 3-methyladenine, requires protein synthesis. Only very limited constitutive activity capable of removing O6-MeGua was detected.
TL;DR: TPA promotion of skin tumors in mice can be modified by application of various prostaglandins or their precursors: PGF2alpha enhances promotion, whereas PGE1 consistently inhibits promotion.
Abstract: TPA promotion of skin tumors in mice can be modified by application of various prostaglandins or their precursors. The effects depend on the particular prostaglandin used: PGF/sub 2..cap alpha../ enhances promotion, whereas PGE/sub 1/ consistently inhibits promotion. Time of application of the prostaglandin with respect to TPA determines whether PGE/sub 2/ enhances or inhibits. Dose-dependent inhibition was observed for arachidonic acid. The prostaglandins alone were unable to elicit tumors in initiated mice.
TL;DR: Perturbation molecular orbital calculations on their presumed ultimate carcinogenic metabolites, the cyclopenta-PAH expoxides, predict that they may have a greater biological hazard than the classic PAHs.
Abstract: Cyclopenta-polycyclic aromatic hydrocarbons (cyclopenta-PAHs) are a group of compounds that have been detected as environmental pollutants. Perturbation molecular orbital (PMO) calculations on their presumed ultimate carcinogenic metabolites, the cyclopenta-PAH expoxides, predict that they may have a greater biological hazard than the classic PAHs.
TL;DR: For a series of mutagens, induction of SCE does not necessarily result from a single specific DNA lesion, therefore, SCE can be considered a qualitative indicator of potential mutagenic events.
Abstract: The mechanism of induction of sister chromatid exchange (SCE) was investigated by treating Chinese hamster V-79 cells with two ethylating and two methylating mutagens at doses, taken from linear response curves, that produced 30 SCE/cell. Concentrations of the DNA alkylation products were measured or calculated at 11 DNA base sites and at the phosphodiester bond. Ethyl methanesulfonate, N-methyl- and N-ethyl-N-nitrosourea produced comparable concentrations (3.3 to 3.5 micromol product/mol DNA phosphate) of O6-alkylguanine. Hence, alkylation at O6 of guanine appears relevant to SCE induction for these mutagens. Since alkylation at O6 of guanine has been positively correlated with mutagenesis in V-79 cells, these findings support the suggestion that SCE and mutagenesis can result from a common DNA lesion. Methyl methanesulfonate (MMS) produced very little O6-methylguanine, but did produce 3-methylthymine and 3-methyladenine, either of which might account for the MMS-induced SCE. Thus, for a series of mutagens, induction of SCE does not necessarily result from a single specific DNA lesion. Therefore, SCE can be considered a qualitative indicator of potential mutagenic events.
TL;DR: Analysis of BP metabolites by high pressure liquid chromatography indicated that epidermal keratinocytes metabolize BP preferentially at non-K-regions such as positions 7, 8, 9 and 10, forming a moderate amount of BP-7,8-dihydrodiol, a precursor of the ultimate metabolite, BP-9,10-epoxide.
Abstract: The metabolism of benzo[a]pyrene (BP) in cultured human epidermal keratinocytes was investigated using thin layer chromatography, high pressure liquid chromatography and cell-mediated mutagenesis assay. Epidermal keratinocytes were obtained from skin of normal subjects and all experiments were performed on primary cultures. Human epidermal keratinocytes were shown to metabolize BP. Analysis of BP metabolites by high pressure liquid chromatography indicated that epidermal keratinocytes metabolize BP preferentially at non-K-regions such as positions 7, 8, 9 and 10, forming a moderate amount of BP-7,8-dihydrodiol, a precursor of the ultimate metabolite, BP-7,8-dihydrodiol-9,10-epoxide. Conjugate formation was examined by treating the medium with beta-glucuronidase and arylsulfatase. No appreciable amount of conjugates was formed by epidermal keratinocytes, except in one culture which gave small peaks eluted in the phenol regions after beta-glucuronidase treatment. The metabolic activity of human epidermal keratinocytes on BP was further demonstrated by a cell-mediated assay, in which V79 Chinese hamster cells were cultured on top of sheets of keratinocytes and treated with BP for 48 h. Mutation of the V79 cells, demonstrated as ouabain resistance, was induced in a dose-related fashion. The extent of induced mutation was higher than that observed using rat embryo cells as the activating layer, although the shape of the dose-response curves was different.
TL;DR: It would appear, therefore, that much of the release of AFB1 from DNA in vivo within the first 24h is probably not through a DNA repair process but through chemical release arising from the positively charged N7-guanine.
Abstract: Following intraperitoneal administration of [3H] aflatoxin B1 (AFB1) to young adult male rats, there is rapid uptake of the carcinogen by the liver, the target organ for carcinogenesis, leading to DNA covalent binding. Acid hydrolysis of this DNA shows that after 2h, the major DNA adduct is trans 8,9-dihydro-8-(7-guanyl)-9-hydroxy AFB1 (AFB1-gua). By 24h after AFB1 administration the major DNA adduct is no longer AFB1-gua but a product with the identical retention time on h.p.l.c. to 8,9-dihydro-8-(N5-formyl-2',5',6' triamino-4' oxo-N5-pyrimidyl)-9-hydroxy AFB1 (AFB1-triamino-Py). 48h after carcinogen administration, only a small amount of AFB1-gua remains and the major product is AFB1-triamino-Py. The half-life of removal of AFB1-gua is 22h, while AFB1-triamino-Py is much more persistent. In vitro incubation studies on DNA isolated from rats treated 2h previously with [3H] AFB1 show that at pH 7.4 AFB1-gua is the major product released from the DNA with some release of 8,9-dihydro-8,9-dihydroxy AFB1, (AFB1-diol). If more extensively reacted AFB1-DNA is used than that obtained from in vivo administration, then the rate of AFB1-diol release is enhanced while that of AFB1-gua is reduced. It would appear, therefore, that much of the release of AFB1 from DNA in vivo within the first 24h is probably not through a DNA repair process but through chemical release arising from the positively charged N7-guanine. There is considerable conversion of AFB1-gua to AFB1-triamino-Py on in vitro incubation of DNA as well as AFB1-gua and AFB1-diol release. By 24h approximately 66% of the bound AFB1 is in the form of AFB1-triamino-Py and after 48h the conversion is complete. The complex pattern of AFB1-release from DNA may have important consequences in both the induction of mutations and in tumour initiation.
TL;DR: The results indicate that DMBA-induced ODC activity may be an important component of the mechanism of DMBA carcinogenesis, and the protective effect of retinoic acid on skin carcinogenesis is not universal; it inhibits skin tumor formation by some agents and not by others.
Abstract: Application of a single large dose (3.6 micromol) or smaller weekly repeated doses (0.2 micromol) of 7,12-dimethylbenz[a]anthracene (DMBA) to the skin of CD-1 mice led to a 20 to 50-fold increase in epidermal ornithine decarboxylase (ODC) (EC 4.1.1.17) activity as well as tumor formation. Retinoic acid (0.17-68 nmol), a potent inhibitor of both the induction of ODC activity and tumor formation by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), failed to inhibit both the induction of ODC activity and tumor formation by DMBA. In contrast, 7,8-benzoflavone (367 nmol), which did not inhibit the induction of ODC activity by TPA, effectively inhibited the induction of ODC activity as well as the formation of skin tumors caused by DMBA. These results indicate that (a) the mechanism of the induction of ODC activity and tumor formation by a complete carcinogen appears to be different from that of the tumor promoter TPA, (b) DMBA-induced ODC activity may be an important component of the mechanism of DMBA carcinogenesis, and (c) the protective effect of retinoic acid on skin carcinogenesis is not universal; it inhibits skin tumor formation by some agents and not by others.
TL;DR: It is concluded that a pigmented xerodermoid cell culture is indistinguishable from the XP variant and the former term is therefore redundant and points to an important role for perturbations in DNA replication in human carcinogenesis.
Abstract: The "pigmented xerodermoid" was previously defined on the basis of mild clinical symptoms that suggested it might be similar to but distinct from xeroderma pigmentosum (XP). XP and pigmented xerodermoid cell cultures were irradiated with ultraviolet light and unscheduled DNA synthesis, strand breakage during repair, chain growth during semiconservative DNA replication with or without caffeine, and the recovery of DNA replication were determined. It is concluded that a pigmented xerodermoid cell culture is indistinguishable from the XP variant and the former term is therefore redundant. The defect common to these cell types appears to be the loss of a gene product that permits normal cells to replicate DNA without interruption at damaged sites (u.v.-induced pyrimidine dimers). The consequence of this loss is that replication forks are blocked more frequently and at lower doses in XP variant cells. The correlation between this defect and high levels of actinic carcinogenesis in these patients points to an important role for perturbations in DNA replication in human carcinogenesis.
TL;DR: Monolayer cultures of human prostatic epithelial cells were exposed to SV40 virus at 35th population doubling and transformed lines were found to have altered morphology, ultrastructure, chromosomes, and growth behavior.
Abstract: Monolayer cultures of human prostatic epithelial cells were exposed to SV40 virus at 35th population doubling. Clones were isolated from infected plates after growth had ceased on the control plates. The nuclei of these clones were virtually all positive for viral T-antigen by immunofluorescence. When the properties of three of these lines were compared to those of normal cells, they were found to have altered morphology, ultrastructure, chromosomes, and growth behavior. All transformed lines had reduced serum dependence and were capable of growing in soft agar. However, their reduced serum dependence was not due to reduced growth factor requirements because each subline's response to growth factors was different.
TL;DR: A mutagenic response was obtained in the Salmonella/microsome reversion test by preincubating sodium nitrite and cimetidine in human gastric juice from untreated individuals, or even by adding nitrite to gastric Juice samples from patients receivingcimetidine.
Abstract: A mutagenic response was obtained in the Salmonella/microsome reversion test by preincubating sodium nitrite and cimetidine in human gastric juice from untreated individuals, or even by adding nitrite to gastric juice samples from patients receiving cimetidine. Both base-pair substitutions (strains TA1535 and TA100) and, though very weakly, also frameshift errors (TA1537, TA1538 and TA98) were induced by such reaction. Mutagenicity was not affected by S-9 mix containing rat liver homogenates, neither in the sense of activation nor of deactivation. The optimal reaction occurred at high equimolar concentrations of the two precursor compounds, under physiological pH and temperature conditions and within a short time of contact. Ascorbic acid was efficient in preventing the formation of mutagenic nitrosoderivative(s).