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Showing papers in "Carcinogenesis in 1989"


Journal ArticleDOI
TL;DR: An antioxidant fraction of Chinese green tea, containing several catechins, has been previously shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin and was shown to have antioxidative activity toward hydrogen peroxide and the superoxide radical.
Abstract: An antioxidant fraction of Chinese green tea (green tea antioxidant; GTA), containing several catechins, has been previously shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin. In the present study, GTA was shown to have antioxidative activity toward hydrogen peroxide (H2O2) and the superoxide radical (O2-). GTA also prevented oxygen radical and H2O2-induced cytotoxicity and inhibition of intercellular communication in cultured B6C3F1 mouse hepatocytes and human keratinocytes (NHEK cells). GTA (0.05-50 micrograms/ml) prevented the killing of hepatocytes (measured by lactate dehydrogenase release) by paraquat (1-10 mM) and glucose oxidase (0.8-40 micrograms/ml) in a concentration-dependent fashion. GTA (50 micrograms/ml) also prevented the inhibition of hepatocyte intercellular communication by paraquat (5 mM), glucose oxidase (0.8 micrograms/ml), and phenobarbital (500 micrograms/ml). In addition, GTA (50 micrograms/ml) prevented the inhibition of intercellular communication in human keratinocytes by TPA (100 ng/ml). Cytotoxicity and inhibition of intercellular communication, two possible mechanisms by which tumor promoters may produce their promoting effects were therefore prevented by GTA. The inhibition of these two effects of pro-oxidant compounds may suggest a mechanism by which GTA inhibits tumor promotion in vivo.

1,895 citations


Journal ArticleDOI
TL;DR: Interindividual differences in the metabolism of chemical carcinogens and repair rates of carcinogen-induced DNA damage reflect acquired and inherited host factors that may influence an individual's risk for development of cancer.
Abstract: Chemical carcinogens are generally activated enzymatically to electrophiles that form covalently bound carcinogen-DNA adducts. Detoxifying enzymes are competing with the activating enzymes for these procarcinogenic chemical substrates. Wide person-to-person variations in these two types of enzymatic activities are found. Repair rates of DNA damage caused by carcinogens also vary among individuals. These interindividual differences in the metabolism of chemical carcinogens and repair rates of carcinogen-induced DNA damage reflect acquired and inherited host factors that may influence an individual's risk for development of cancer.

208 citations


Journal ArticleDOI
TL;DR: Green tea polyphenols (GTP) were evaluated as an anti-initiating agent against the skin tumorigenicity induced by polycyclic aromatic hydrocarbons (PAHs) in mice and suggest that GTP has substantial anti-skin-tumor- initiating activity against PAHs.
Abstract: Green tea is a popular beverage in China and Asia and has been shown to possess antipyretic, diuretic and several other pharmacological activities. The major constituents of green tea are polyphenols which have been found to possess antioxidant and antimutagenic properties. In this study green tea polyphenols (GTP) were evaluated as an anti-initiating agent against the skin tumorigenicity induced by polycyclic aromatic hydrocarbons (PAHs) in mice. In a complete skin tumorigenesis protocol using 3-methylcholanthrene the topical application of GTP to female BALB/c mice resulted in substantial protection against the onset and subsequent development of tumors. In the two-stage skin tumorigenesis protocol using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiating agent followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate; (TPA) as tumor promoter, topical application of GTP to female SENCAR mice afforded significant protection against skin tumorigenicity. Oral feeding of GTP in drinking water to female SENCAR mice also protected against skin tumorigenesis in DMBA - TPA-treated animals. GTP when administered topically or orally significantly inhibited PAH - DNA adduct formation in epidermis after topical application of [3H]benzo[a]pyrene or [3H]DMBA. Our results suggest that GTP has substantial anti-skin-tumor-initiating activity against PAHs and could prove useful in protecting against some forms of human cancer.

184 citations


Journal ArticleDOI
TL;DR: The data show that TE671 and RD are derivatives of the same cell line and it is concluded that the properties of the TE671 line should be ascribed to rhabdomyosarcoma rather than medulloblastoma cells.
Abstract: The cell line TE671 has been widely used as a model of human medulloblastoma. In the present study we have demonstrated that transfection of DNA from this cell line into NIH 3T3 cells reveals the presence of an activated N-ras gene. Using oligonucleotide probes we have shown that the N-ras gene is activated by a point mutation at the third base of codon 61 resulting in the substitution of histidine for glutamine in the p21 ras gene product. We noted that this relatively uncommon activating mutation is also present in the human rhabdomyosarcoma cell line RD. Based on this finding and on the observation that several of the phenotypic characteristics of TE671, such as the presence of muscle-type nicotinic acetylcholine receptors and the intermediate filament protein desmin, are suggestive of myoid origin we investigated the possible identity of these two cell lines. Cytogenetic analysis revealed the presence of marker chromosomes common to both TE671 and RD. DNA fingerprinting using both locus specific and multilocus core probes showed indistinguishable band patterns in the two cell lines. Taken together our data show that TE671 and RD are derivatives of the same cell line and we conclude that the properties of the TE671 line should be ascribed to rhabdomyosarcoma rather than medulloblastoma cells.

179 citations


Journal ArticleDOI
TL;DR: The monocyclic monoterpenoid d-limonene and its source, orange oil, were found to prevent rat mammary carcinomas induced by the direct-acting carcinogen nitrosomethylurea.
Abstract: The monocyclic monoterpenoid d-limonene and its source, orange oil, were found to prevent rat mammary carcinomas induced by the direct-acting carcinogen nitrosomethylurea. This chemopreventive effect was limited to the promotion/progression stage in this carcinogenesis model.

155 citations


Journal ArticleDOI
TL;DR: It is proposed that much of the chemopreventive action of retinoids can be explained by the enhanced junctional communication of growth regulatory signals.
Abstract: Retinoids that cause inhibition of methylcholanthrene-induced neoplastic transformation of C3H/10T1/2 cells enhance gap-junctional communication in carcinogen-initiated cells. Dose-response studies using retinoids of diverse structures and potency demonstrated a good correlation between these two events. Junctional permeability was enhanced by retinol and tetrahydrotetramethylnaphthalenyl propenylbenzoic acid (TTNPB) at concentrations from 10(-10) to 10(-6) M, and by retinoic acid between 10(-8) and 10(-6) M, the same concentrations that inhibited neoplastic transformation. Retinoic acid inhibited permeability at 10(-10) M, at which concentration transformation was enhanced. Retinoids caused similar alteration sin communication in parental 10T1/2 cells. Communication between initiated and 10T1/2 cells was not influenced by TTNPB. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited junctional communication in initiated cells, in 10T1/2 cells and between these two cell lines. After repeated exposure of 10T1/2 cells to TPA only retinoid-enhanced communication was blocked; in contrast, basal communication became refractory. It is proposed that much of the chemopreventive action of retinoids can be explained by the enhanced junctional communication of growth regulatory signals.

154 citations


Journal ArticleDOI
TL;DR: A useful bioassay is established for identification of compounds that can modify NNK-induced lung tumorigenesis in female A/J mice by the tobacco-specific nitrosamine 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanone (NNK).
Abstract: This paper describes the development of a relatively rapid single-dose model for induction of lung adenomas in female A/J mice by the tobacco-specific nitrosamine 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanone (NNK). Mice maintained on AIN-76A semi-synthetic diet were given a single i.p. dose of 2.5, 5 or 10 mumol NNK in saline and killed 3-7 months later. Maximum lung tumor induction, measured by lung tumors per mouse (tumor multiplicity), occurred in 3.5 months. There was no significant increase in tumor multiplicity between 3.5 and 7 months. Four months after treatment, numbers of lung tumors per mouse were 11.9 +/- 1.0 (10 mumol NNK), 3.6 +/- 0.4 (5 mumol), 0.9 +/- 0.4 (2.5 mumol) and 0.07 +/- 0.1 (saline). Lung tumor multiplicity in mice treated with a single dose of 10 mumol NNK and maintained on AIN-76A diet was significantly higher (8.3 +/- 0.5) than in mice treated with NNK and maintained on NIH-07 diet (2.5 +/- 0.3). The results of this study establish a useful bioassay for identification of compounds that can modify NNK-induced lung tumorigenesis.

149 citations


Journal ArticleDOI
TL;DR: From data, a mechanism for the DNA strand scission reaction of quercetin and related flavonoids is proposed andCu(I) was shown to be an essential intermediate by using the Cu(I)-sequestering reagents, neoc uproine and bathocuproine.
Abstract: The naturally occurring flavonoid, quercetin, in the presence of Cu(II) and molecular oxygen caused breakage of calf thymus DNA, supercoiled pBR322 plasmid DNA and single-stranded M13 phage DNA. In the case of the plasmid, the product(s) were relaxed circles or a mixture of these and linear molecules depending upon the conditions. For the breakage reaction, Cu(II) could be replaced by Fe(III) but not by other ions tested [Fe(II), Co(II), Ni(II) and Ca(II)]. Structurally related flavonoids, rutin, galangin, apigenin and fisetin, were ineffective or less effective than quercetin in causing DNA breakage. In the case of the quercetin--Cu(II) reaction, Cu(I) was shown to be an essential intermediate by using the Cu(I)-sequestering reagents, neocuproine and bathocuproine. By using Job plots we established that, in the absence of DNA, five Cu(II) ions can be reduced by one quercetin molecule; in contrast, two ions were reduced per quercetin molecule in the DNA breakage reaction. Equally neocuproine inhibited the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, iodide, mannitol, formate and catalase (the inhibition was complete in the last case). From these data we propose a mechanism for the DNA strand scission reaction of quercetin and related flavonoids.

141 citations


Journal ArticleDOI
TL;DR: Normal and neoplastic keratinocytes differ in the level of Cai under low calcium conditions and in their response to elevated external calcium, which may be important in determining their potential for terminal differentiation.
Abstract: Normal keratinocytes proliferate when cultured in medium with 002-010 mM calcium and terminally differentiate when medium calcium is increased to greater than 01 mM In contrast, neoplastic keratinocyte cell lines maintain the potential for continued cell renewal and survive when external calcium is increased In order to determine whether elevation of extracellular calcium produced changes in intracellular free calcium (Cai) levels, Cai was measured in individual living keratinocytes by use of the fluorescent calcium probe fura-2 Most normal keratinocytes responded to increased extracellular calcium by a gradual 2- to 3-fold increase in Cai lasting for at least 28 min A subpopulation displayed a sharp peak of Cai at 2 min In contrast, the Cai level in neoplastic cells in either low or high calcium medium was 2- to 3-fold higher than that in normal cells, and all cells in the population showed a transient 4- to 9-fold elevation of Cai 2 min after external calcium was increased Thus normal and neoplastic keratinocytes differ in the level of Cai under low calcium conditions and in their response to elevated external calcium The regulation of Cai in keratinocytes may be important in determining their potential for terminal differentiation

137 citations


Journal ArticleDOI
TL;DR: Evaluation of a system screening for intrachromosomal recombination that results in genome rearrangement has been constructed for potential use as a short-term test in the yeast Saccharomyces cerevisiae shows that it is readily inducible by a variety of mutagenic as well as non-readily inducable carcinogens.
Abstract: To identify environmental carcinogens there is a need for inexpensive and reliable short-term tests that can be used to predict the carcinogenic potential of any given substance with high accuracy. The Ames assay, which is based on the induction of mutations in Salmonella typhimurium, is the most extensively used short-term test but certain human or animal carcinogens exist that are persistently undetectable as mutagens with the Ames assay or with other short-term tests. There is a need for a short-term test to detect those carcinogens that are missed by the Ames assay. Carcinogenesis is in many cases associated with genome rearrangement. Because of this association a system screening for intrachromosomal recombination that results in genome rearrangement has been constructed for potential use as a short-term test in the yeast Saccharomyces cerevisiae. Evaluation of this recombination system shows that it is readily inducible by a variety of mutagenic as well as non-readily inducible by a variety of mutagenic as well as non-mutagenic carcinogens, including carcinogens that are not detectable by the Ames assay or by various other short-term tests, such as safrole, urethane, ethionine, auramine, methylene chloride, carbon tetrachloride, cadmium sulfate, aniline, dimethylhydrazine, aminotriazole, acetamide, thiourea and DDE. The present report shows the data for these as well as for additional agents, their response profiles with different concentrations of the agents and the protocol for the DEL system.

127 citations


Journal ArticleDOI
TL;DR: Tests have been carried out with several plant flavonoids to detect their ability to suppress mutagenesis in Salmonella typhimurium strain TA100 NR induced by the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine.
Abstract: Tests have been carried out with several plant flavonoids to detect their ability to suppress mutagenesis in Salmonella typhimurium strain TA100 NR induced by the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Among the most effective flavonoids are the isoflavone, biochanin A, the flavanone glycoside, naringin, and its aglycone, naringenin, and several flavonols, e.g. morin, fisetin, kaempferol, gossypetin and quercetin, including a flavonol glycoside, rutin. In particular, naringin possesses exceptional antimutagenic activity, in as much as, less than half the equimolar amount can reduce the mutagenic potency of this carcinogen by 50%. These flavonoids appear to act either by preventing passage of the carcinogen into bacterial cells or by altering some cellular processes.

Journal ArticleDOI
TL;DR: Surprisingly, Ins, an in vitro growth promoting agent also caused a significant (P less than 0.001) suppression of LIC and InsP6 +/- Ins also showed a concomitant reduction in the mitotic rate in the non-neoplastic epithelium.
Abstract: In previous studies, we have shown that inositol hexaphosphate (InsP6), a constituent of cereal diet, inhibited azoxymethane-induced experimental large intestinal cancer (LIC) in Fischer 344 rats. We now report a similar antineoplastic action of InsP6 in CD-1 mice injected with 1,2-dimethylhydrazine (DMH). We had hypothesized that InsP6 may bring about this effect by undergoing dephosphorylation to lower phosphorylated forms; the ready availability of Ins, to react with phosphates, may increase the total amount of the lower phosphorylated Ins and potentiate the action of InsP6. LIC induced by DMH (15 mg/kg/week x 13) in mice given a mixture of 1% InsP6 + 1% Ins show a significant reduction (P less than 0.005) in LIC prevalence over InsP6 treatment. Surprisingly, Ins, an in vitro growth promoting agent also caused a significant (P less than 0.001) suppression of LIC. InsP6 +/- Ins also showed a concomitant reduction in the mitotic rate in the non-neoplastic epithelium. Body weight data did not suggest any overt toxic effect of long-term administration of InsP6, Ins or InsP6 + Ins. Since InsP6 is antineoplastic in two species of experimental animals, it should, in combination with Ins, be considered in our strategies for prevention of large intestinal cancer.

Journal ArticleDOI
TL;DR: Using the wing hair spot test, it is found that the formation of mutant hairs in adult flies as a result of feeding them with Trp-P-2 in their larval stage was efficiently inhibited by coadministration of chlorophyll.
Abstract: Genotoxicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) on Drosophila was suppressed by chlorophyll. Using the wing hair spot test, we found that the formation of mutant hairs in adult flies as a result of feeding them with Trp-P-2 in their larval stage was efficiently inhibited by coadministration of chlorophyll. The decrease in the spot frequencies was dependent on the dose of chlorophyll, and at the highest dose used, where the ratio in weight of Trp-P-2 to chlorophyll was 1:80, a complete prevention of the small single-spot formation was observed. A similar inhibitory effect was detected for chlorophyllin, the chromophore of chlorophyll. In the studies to investigate the mechanism of inhibition, we observed that the mutagenicity of 3-hydroxy-amino-1-methyl-5H-pyrido[4,3-b]indole [Trp-P-2(NHOH)], the metabolically activated form of Trp-P-2, in Salmonella typhimurium TA98 was suppressed effectively with chlorophyll and chlorophyllin. We also found that chlorophyll and chlorophyllin can produce complexes with Trp-P-2 and Trp-P-2(NHOH). A straightforward mechanism of these inhibitions is that Trp-P-2 [and Trp-P-2(NHOH)] becomes no longer available to organisms on forming the chlorophyll complex.

Journal ArticleDOI
TL;DR: Data show for the first time that the magnitude and time course of lipofuscin deposition, induction of lysosomal enzymes and conjugated diene accumulation, is correlated closely with the degree of carcinogenicity of Wy-14,643.
Abstract: Quantitative comparisons of the time course of biochemical and morphological changes induced by peroxisome proliferators resulting in low and high incidences of hepatic cancer have not been conducted previously under bioassay conditions. [4-Chloro-6-(2,3-xylidino)-2-pyrimidyl-thio]acetic acid (Wy-14,643) at 0.1% in the diet produced a much higher incidence of hepatic cancer in male rats than 1.2% di(2-ethylhexyl)phthalate (DEHP) in the diet. Both diets, however, caused similar degrees of peroxisome proliferation. To investigate this difference in carcinogenicity, H2O2-detoxification mechanisms and indices of oxidative damage were evaluated in male F-344 rats fed 1.2% DEHP or 0.1% Wy-14,643 for up to one year. DEHP or Wy-14,643 treatment increased hepatic catalase activity approximately 25% from 8 to 365 days. DEHP or Wy-14,643 treatment decreased hepatic glutathione peroxidase activity by 50% from 8 to 365 days. Glutathione concentrations were not affected by 151 days of DEHP or Wy-14,643 feeding. The similar effects of DEHP and Wy on H2O2 detoxification enzymes and glutathione concentrations suggests that these factors are not responsible for the widely different carcinogenicities of Wy-14,643 and DEHP. Hepatic vitamin E concentrations were 50% lower in rats receiving Wy-14,643 for 151 days as compared to rats fed DEHP or control diets. Lipofuscin, which was contained within lysosomes, was increased 3-fold after 39 days of DEHP and remained at this level up to 365 days of treatment. In comparison, lipofuscin was increased 4-fold after 18 days of Wy-14,643 and continued to accumulate in a linear manner reaching values 30-fold over controls after 365 days of treatment. DEHP treatment for 39-365 days increased the activities of the lysosomal enzymes alpha-fucosidase, beta-galactosidase and N-acetylglucosaminidase 50-100%. The same enzyme activities were increased approximately 4-fold after 39-365 days of Wy-14,643. Lysosomal cathepsin B activity was unchanged by DEHP but doubled by 151 and 365 days of Wy-14,643. Acid phosphatase activity was unchanged by DEHP but increased by 50% after 151 and 365 days of Wy-14,643. In addition, conjugated dienes were increased (approximately 45%) only in rats receiving Wy-14,643 for 151 and 365 days. These data show for the first time that the magnitude and time course of lipofuscin deposition, induction of lysosomal enzymes and conjugated diene accumulation, is correlated closely with the degree of carcinogenicity. Wy-14,643-induced decreases in hepatic vitamin E concentrations could contribute to the observed accumulation of conjugated dienes at later time points.(ABSTRACT TRUNCATED AT 400 WORDS)

Journal ArticleDOI
TL;DR: PGF2 alpha, appearing as one distinct biosynthetic wave 3-4 h after TPA application seems to be critically involved in the conversion steps since inhibition of its accumulation by indomethacin led to an inhibition of tumor formation and the inhibitory effect of indometHacin on papilloma formation was reversed by PGF2alpha treatment concomitant with RPA.
Abstract: When applied to NMRI mouse skin in vivo, phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12-O-retinoylphorbol-13-acetate (RPA) have been found to induce two early waves of prostaglandin E2 (PGE2) synthesis after 15 and 90 min and a delayed accumulation of prostaglandin F2 alpha (PGF2 alpha) after 2 h. With respect to PGF2 alpha formation different activities of both agents were observed, in that TPA but not RPA induced additional PGF2 alpha waves after 4 and 17 h. Functionally, PGE2 was previously shown to be an endogenous mediator of the TPA- or RPA-induced epidermal hyperproliferation and hyperplasia. A functional role of PGF2 alpha could be attributed to the post-initiation stages of tumor development in initiated mouse skin, i.e. the conversion stage (stage I of tumor promotion) elicited by two TPA applications and the promotion stage (stage II of promotion) brought about repetitive RPA treatments. PGF2 alpha, appearing as one distinct biosynthetic wave 3-4 h after TPA application seems to be critically involved in the conversion steps since (i) inhibition of its accumulation by indomethacin led to an inhibition of tumor formation, (ii) the inhibitory effect of indomethacin could be reversed by PGF2 alpha and (iii) RPA was not able to give rise to the accumulation of PGF2 alpha 4 h after application as obtained by TPA treatment. Moreover, RPA-induced promotion of DMBA- and TPA-treated mouse skin was inhibited by indomethacin. The inhibitory effect of indomethacin on papilloma formation was again reversed by PGF2 alpha treatment concomitant with RPA.

Journal ArticleDOI
TL;DR: A general trend of increasing inhibition of NNK-induced lung neoplasia by arylalkyl isothiocyanates with increasing alkyl chain length is demonstrated.
Abstract: Six homologous arylakyl isothiocyanates were evaluated for their abilities to inhibit pulmonary adenomas induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. Four consecutive daily doses (5 mumol/mouse) of phenyl isothiocyanate (PITC), benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), 4-phenylbutyl isothiocyanate (PBITC), 4-oxo-4-(3-pyridyl)-butyl isothiocyanate (OPBITC) and corn oil were administered to mice by gavage. Two hours following the final dosing, mice were administered saline or 10 mumol of NNK in saline i.p. Pulmonary adenomas were counted at 16 weeks after NNK administration. The mice administered only corn oil prior to NNK developed an average multiplicity of 9.2 tumors/mouse. Pretreatment with PITC, BITC and OPBITC had no significant effects on NNK-induced lung neoplasia. However, PEITC pretreatment resulted in a 64% reduction of lung tumor multiplicity, but did not affect the percentage of mice that developed tumors. Both PPITC and PBITC decreased tumor multiplicity by 96% and the percentage of tumor-bearing animals by greater than 60%. These results, in conjunction with our previous work, demonstrate a general trend of increasing inhibition of NNK-induced lung neoplasia by arylalkyl isothiocyanates with increasing alkyl chain length. This study also demonstrates the remarkable inhibitory activities of PPITC and PBITC, two isothiocyanates that had not previously been tested as chemopreventive agents.

Journal ArticleDOI
TL;DR: The method is applicable to the analysis of DNA base damage caused by major endogenous processes relevant to aging, such as deamination, methylation and oxidation, and may be potentially useful for studies on carcinogenesis and in studies on the epidemiology of cancer.
Abstract: A method for the detection of rare adducts in DNA has been developed by combining the resolution of high-performance liquid chromatography with the specificity and sensitivity of electrochemical detection. Many adducts are electrochemically active, while the normal bases, except for guanine, are not. Enzymatic hydrolysis of DNA is used to obtain the deoxynucleosides for analysis, or where appropriate, acid hydrolysis or thermal depurination of DNA is used to free the adduct base for analysis. Various types of DNA damage have been induced by in vitro exposure of DNA to acrolein, dimethyl sulfate, sodium nitrite, ascorbate/Cu2+ and gamma-irradiation. Several adducts are detected at a level of one adduct in 10(5)-10(6) normal bases in micrograms of DNA. The method is also useful for measuring O6-methylguanine (O6MeGua) in DNA from rats treated with N-nitrosodimethylamine and 8-hydroxydeoxyguanosine (oh8dG), and O6-MeGua in DNA from bacteria treated with hydrogen peroxide and dimethyl sulfate. oh8dG, which appears to be the most suitable marker for measuring the steady-state level of oxidative DNA damage, can be measured at fmol levels in DNA from normal rat tissues. The method is applicable to the analysis of DNA base damage caused by major endogenous processes relevant to aging, such as deamination, methylation and oxidation. The analysis of DNA adducts with this simple assay also may be potentially useful for studies on carcinogenesis and as a tool in studies on the epidemiology of cancer.

Journal ArticleDOI
TL;DR: The results suggest that the early GST-P positive populations could be the precursor for preneoplastic foci and nodules in rats soon after treatment with hepatocarcinogens.
Abstract: The single cells positive for placental glutathione S-transferase (GST-P), detectable in livers of rats soon after treatment with hepatocarcinogens, are possible 'initiated cells', the hypothesis tested in the present series of experiments. No low dose threshold was observed in male Sprague-Dawley rats at different single doses of diethylnitrosamine (DEN) although a plateau was reached between 160 and 200 mg/kg body weight. At the latter single dose approximately 12,400 positive cells/cm3 were observed immunohistochemically in rat livers after one week, the numbers then decreasing to week 8 and thereafter rising again. In the early stages single cells predominated but with time a gradual increase in mini-foci and larger lesions became evident. Application of selection pressure (feeding of diet containing 0.02% 2-AAF plus partial hepatectomy) to rats 2-24 weeks after single DEN-treatment resulted in the formation of large foci positive for GST-P, especially in the early stages, the growth response being less pronounced with time. The number of foci, on the other hand, was correlated with the number of single cells/mini-foci detected in hepatectomy tissue of the same individuals. These results suggest that the early GST-P positive populations could be the precursor for preneoplastic foci and nodules.

Journal ArticleDOI
TL;DR: High performance liquid chromatography combined with synchronous fluorescence spectroscopy provides a highly specific method for the detection of covalently bound BP residues in both human hemoglobin and DNA.
Abstract: Highly specific methods are required to detect and quantitate carcinogen-macromolecular adducts in humans who are exposed to complex mixtures of chemical carcinogens. High performance liquid chromatography and fluorescence spectroscopy have been used successfully to detect and identify residues of benzo[a]pyrene-7,10/8,9-tetrahydrotetrol (BP-7,10/8,9-tetrol) that were released upon mild acid hydrolysis of human DNA or hemoglobin. Synchronous fluorescence spectroscopy data indicate that levels of benzo[a]pyrene-diol-epoxide-DNA (BPDE-DNA) adducts as high as 1.54 fmol BPDE/micrograms DNA are formed (1 adduct in 5 million nucleotides) in peripheral blood lymphocytes of coke-oven workers; these data were subsequently corroborated by gas chromatography/mass spectroscopy single ion monitoring analysis (m/z 404+). Additionally, among lung cancer patients, 5 samples of tumor DNA were found to be negative and 1 of 4 samples of corresponding lung tissue was found to be positive. Extraction and purification of BP-7,10/8,9-tetrol from the hemoglobin of smokers suggested levels of bound carcinogen in excess of 1 ng BPDE/gm of hemoglobin. High performance liquid chromatography combined with synchronous fluorescence spectroscopy provides a highly specific method for the detection of covalently bound BP residues in both human hemoglobin and DNA.

Journal ArticleDOI
TL;DR: It is postulate that InsP6 may exert its antineoplastic effect by way of regulating cellular proliferation even after effective carcinogenic stimuli and thus may be an important candidate for chemointervention.
Abstract: A treatment regimen of 2% Na-InsP6 in drinking water was effective in significantly reducing large intestinal cancer in F344 rats even when the treatment was begun 5 months after carcinogenic induction with azoxymethane (AOM 8 mg/kg/wk X 6). Compared to untreated (AOM-only) rats, animals on InsP6 had 27% fewer tumors (P less than 0.02). The tumors were approximately two-thirds smaller in size (P less than 0.01) and percentage mitotic rate in the non-neoplastic epithelium was less than half (1.0 +/- 0.1, compared to 2.3 +/- 0.2 of AOM-only animals, significant at P less than 0.001). We postulate that InsP6 may exert its antineoplastic effect by way of regulating cellular proliferation even after effective carcinogenic stimuli and thus may be an important candidate for chemointervention.

Journal ArticleDOI
TL;DR: The results show that the availability of three different types of 32P-postlabeled derivatives for the same adduct aids in the analysis and chromatographic characterization of DNA adducts from diverse exogenous and endogenous sources.
Abstract: A new sensitive 32P-postlabeling assay for DNA adducts has been developed in which DNA is hydrolyzed initially by nuclease P1 and prostatic acid phosphatase instead of micrococcal nuclease and spleen phosphodiesterase as employed in previous postlabeling procedures. When DNA containing bulky adducts, X1, X2, .....Xn, is digested with nuclease P1 at pH 5, normal nucleotides are released as 5'-monophosphates, pN, while adducts are excised as 5'-phosphorylated dinucleotides, pXipN, because internucleotide linkages on the 3' side of X resist attack by nuclease P1. Addition of prostatic acid phosphatase to such a digest results in 5'-dephosphorylation of the nucleotides to normal nucleosides, N, and adducted dinucleotides, XipN, carrying a 5'-terminal free hydroxyl group. The dinucleotides but not nucleosides are converted to 5'-32P-labeled dinucleotides, [32P]pXipN, by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. Upon mapping on polyethyleneimine--cellulose anion-exchange TLC, the labeled dinucleotide adducts produce characteristic autoradiographic fingerprints. Alternatively, they are further digested with snake venom phosphodiesterase to yield 5'-monophosphates, [32P]pXi and pN. TLC profiles of the monophosphate adducts are distinct from those of the dinucleotides. These reactions provide the basis of the new 32P-postlabeling scheme, which is compared in this paper with a previously reported protocol yielding adducts in the form of 5'-32P-labeled 3',5'-bisphosphates, [32P]pXip. The results show that the availability of three different types of 32P-postlabeled derivatives for the same adduct aids in the analysis and chromatographic characterization of DNA adducts from diverse exogenous and endogenous sources.

Journal ArticleDOI
TL;DR: The trout model is employed in a combined DNA binding/tumor dose-response protocol and the first direct evidence of pure anti-initiating activity by a natural anti-carcinogen found in human diet is found, particularly for 'ambivalent modulators' which exhibit both inhibitory and promotional activity.
Abstract: A number of recent studies have described inhibitor-mediated reductions in the covalent DNA binding and tumorigenicity of various carcinogens, in species such as rats, mice and rainbow trout (Salmo gairdneri). Since inhibitory effects have, in most cases, been reported after testing at one carcinogen and one inhibitor level only, the detailed relationships between carcinogen dose, inhibitor dose, in vivo DNA binding and final tumor response are not well understood in any species. To determine these relationships we have employed the trout model in a combined DNA binding/tumor dose-response protocol using approximately 10,000 animals. Trout were pretreated with one of five different dose-levels of indole-3-carbinol (I3C), a naturally occurring anti-carcinogen found in cruciferous vegetables such as broccoli and cabbage. After 4 weeks, animals received the same dietary level of I3C for a further 2 weeks together with [3H]aflatoxin B1 (AFB1) in the dose-range 10-320 p.p.b. From tanks containing 150 animals (three tanks per I3C-AFB1 dose-point), 15 fish were selected at random in order to assess hepatic AFB1-DNA binding levels. Remaining animals were returned to control diet for determination of tumor response at 12 months. Linear increases in DNA binding occurred with dose of AFB1 at each I3C dose-level. Successive increases in I3C dose gave dose-related decreases in AFB1-DNA binding, resulting in a series of curves of decreasing slope. Shifts in DNA-binding slopes were compared quantitatively with horizontal displacements towards higher carcinogen dose in corresponding tumor dose-response curves. At I3C doses of less than or equal to 2000 p.p.m., the inhibitor-altered tumor response was predicted precisely by changes in dose received (DNA adducts formed) in the target organ. These data constitute the first direct evidence of pure anti-initiating activity by a natural anti-carcinogen found in human diet, where all animals were treated at the same time and under identical conditions of exposure in both DNA binding and tumor studies. The data are discussed further in view of (i) their implications for DNA binding-carcinogenicity correlations and the concept of 'molecular dosimetry', and (ii) limitations in the current database on anti-carcinogenesis as regards in vivo potency information, particularly for 'ambivalent modulators' which exhibit both inhibitory and promotional activity.

Journal ArticleDOI
TL;DR: Results indicate that significant in vivo formation of nitrite and volatile N-nitroso compounds occurs in the urinary bladder of bilharzia patients and this may be an oetiological factor in the induction of bilHarzial bladder cancer associated with S. haematobium infection.
Abstract: Saliva and 24-h urine samples were collected from male Schistosomiasis (bilharzia) patients with S. haematobium infection and possible concurrent S. mansoni infection without diagnosed bladder cancer (n = 27), bilharzia patients with diagnosed bladder cancer (n = 23) as well as a comparative control group (n = 27) of healthy Egyptian volunteers with no current bilharzia infection and/or bacterial urinary tract infections from the Nile Delta area of Egypt. Saliva samples were analysed for the presence of nitrate and nitrite; urine samples were analysed for the presence of nitrate, nitrite, volatile and non-volatile N-nitroso compounds. Bilharzia patients prior to, and after, diagnosed bladder cancer regularly excreted free nitrite as well as volatile nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopiperidine and N-nitrosopyrrolidine) in addition to which elevated concentrations of non-volatile N-nitrosamino acids (N-nitrosoproline, N-nitrososarcosine, N-nitrosothiazolidine-4-carboxylic acid and its 2-methyl derivative) were also present. Total urinary excretion of volatile N-nitroso compounds (0.32 +/- 0.64 micrograms/day; mean +/- SD) and non-volatile N-nitroso compounds (31.20 +/- 22.07 micrograms/day) was observed in the Egyptian control group. Significantly higher concentrations were found in bilharzia patients: 3.47 +/- 6.42 (P less than 0.05) and 62.91 +/- 21.96 (P less than 0.05); as well as in bilharzia patients with diagnosed bladder cancer: 1.71 +/- 1.96 (P less than 0.02) and 44.94 +/- 7.31 respectively. Free nitrite was found in the urine of two volunteers in the Egyptian control group (1.7 and 3.0 micrograms/day), urinary nitrite was significantly increased in bilharzia patients (5.18 +/- 9.11 micrograms/day, P less than 0.02) and in bladder cancer patients (1.75 +/- 2.81 micrograms/day, P less than 0.05). Nitrate concentrations were elevated from 139.3 +/- 82.2 in the control group to 143.6 +/- 136.3 and 175 +/- 190 in the bilharzia and bladder cancer groups respectively. These results indicate that significant in vivo formation of nitrite and volatile N-nitroso compounds occurs in the urinary bladder of bilharzia patients and this may be an oetiological factor in the induction of bilharzial bladder cancer associated with S. haematobium infection.

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TL;DR: In vivo treatment of mice with InsP6 enhances baseline NK activity and reverses DMH-induced depressed NK activity with an inverse correlation, suggesting yet another important role of inositol phosphates in the regulation of cellular activity.
Abstract: In recent studies, we have demonstrated that inositol hexaphosphate (InsP6) inhibits experimental colon carcinogenesis. Since natural killer (NK) cells are involved in tumor cell destruction, we investigated the effect of InsP6 on murine NK cell activity. We show that; (i) 1,2-dimethylhydrazine (DMH), a colon carcinogen, depresses NK activity; (ii) in vivo treatment of mice with InsP6 enhances baseline NK activity and reverses DMH-induced depressed NK activity with an inverse correlation (r = -0.9811) with tumor incidence, (iii) short-term in vitro treatment of spleen cells and NK-enriched fraction with InsP6 also enhances NK cytotoxicity in a dose-dependent manner, (iv) inositol potentiates the action of InsP6. Our data suggest yet another important role of inositol phosphates in the regulation of cellular activity.

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TL;DR: The results indicate that neutrophils cause prolonged DNA damage in neighboring cells and indicate that although H2O2 produced in the oxidative burst is an essential mediator of the damage observed, additional reactive oxygen intermediates including the superoxide anion are also implicated.
Abstract: We have measured the capacity of highly-purified, paraffin oil-elicited neutrophils to induce DNA single-strand breaks in a newly established plasmacytoma cell line, RIMPC 2304, which was induced by a retrovirus containing the c-myc and V-Ha-ras oncogenes. This cell line effectively repairs DNA damage induced by gamma-irradiation. DNA damage induced by neutrophils was correlated with the oxidative burst of the neutrophils. The levels of superoxide anion, H2O2, and HOCl produced after stimulation of the neutrophils (6 X 10(5)/cm3) with the tumor promoter phorbol myristate acetate (100 nM) were 33.8 microM, 12.8 microM and 1.7 microM respectively in 15 min, and 98 microM, 20 microM and 8.7 microM respectively in 90 min. The results of alkaline elution experiments revealed that when the same concentration of neutrophils was co-incubated for 15 min in serum-free medium with an equal number of radioactively labeled RIMPC 2304 cells, the latter incurred a level of damage that approximated that caused by 300 rad equivalents of gamma-irradiation or by a 1-min treatment with 20 microM H2O2 at 37 degrees C. Damage from neutrophils was coincident with the oxidative burst; it was induced rapidly (within 5 min) but remained high for more than 90 min. The level of damage achieved was dependent upon the ratio of neutrophils: target cells and was clearly detectable at ratios as low as 0.25:1. Induction of single-strand breaks was completely inhibited by catalase and partially inhibited by superoxide dismutase, mannitol, and reduced glutathione but not by Na azide. Addition of the non-steroidal anti-inflammatory drug indomethacin either enhanced (at 50 microM) or had no effect (at 2 microM) on the damage detected. Finally, repair of strand breaks induced by neutrophils was significantly slower (half-time approximately 10 min) than that observed for repair of similar levels of damage induced by H2O2 or gamma-irradiation (half-times approximately 3 min, each). The results indicate that neutrophils cause prolonged DNA damage in neighboring cells. Moreover, they indicate that although H2O2 produced in the oxidative burst is an essential mediator of the damage observed, additional reactive oxygen intermediates including the superoxide anion are also implicated. The data are discussed in relation to the possible role of neutrophils in chronic inflammation and in pristane-induced plasmacytoma formation in mice.

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TL;DR: It is concluded that creatine and free amino acids are the main reactants of the mutagen-forming reactions that occur during frying of meat.
Abstract: Creatine or one of 15 amino acids were mixed with minced pork before broiling at 200 degrees C. Total mutagenic activity and reversed-phase HPLC-separated mutagenicity profiles were determined for the crust and pan residue of all samples and also in the aerosol fraction of the smoke formed during cooking of the creatine-fortified samples. Addition of 5% (w/w) creatine increased the total mutagenicity 4-fold without changing the mutagenicity profile of either crust, pan residue or aerosol. Amino acid addition (1% w/w) increased the total mutagenicity between 1.5 (lysine) and 43 times (threonine). In most cases the mutagenicity profiles of crust and pan residues were changed by amino acid addition. Dry-heated mixtures of amino acids and creatine were all mutagenic with a 250-fold range between the amino acids. The production of known food mutagens in these mixtures was analyzed by LC-MS of HPLC-fractionated mutagenic peaks. Serine, threonine, phenylalanine, alanine, leucine and tyrosine were all shown to give rise to one of the known food mutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-trimethylimidazopyridine (TMIP). Lyophilized and subsequently fried meat patties and a heated powder of lyophilized meat juice were both mutagenic, with mutagenicity profiles similar to the regular meat crust, showing that water is not a prerequisite for mutagen formation in meat. MeIQx, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-di-MeIQx) and PhIP were shown, by LC-MS, to be present in the dry-heated meat juice. It is concluded that creatine and free amino acids are the main reactants of the mutagen-forming reactions that occur during frying of meat. Creatine is probably a necessary part of all of these reactions; what specific compounds are formed in each case therefore depends upon the levels in the meat of certain free amino acids and their interactions with other, as yet unknown, compounds in the meat.

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TL;DR: It is suggested that S-PMA can be regarded as a useful indicator for monitoring individuals and collectives exposed to benzene at levels even less than 1 p.p.m and the correlation between benzene uptake and the excretion of the urinary metabolites was significant.
Abstract: In an inhalation study rats were exposed to different doses of benzene, ranging from 1 to 500 p.p.m. The urine was sampled during the inhalation period of 8 h and for 24 h after exposure. S-Phenylmercapturic acid (S-PMA) in the urine was determined by amino acid analysis. Phenol was measured by gas chromatography/mass spectrometry. In both cases the correlation between benzene uptake and the excretion of the urinary metabolites was significant at the level of P = 0.01. The same significant correlation (P = 0.01) was demonstrable after i.p. administration of benzene at doses between 0.7 and 140.0 microliters/kg body weight. In the case of two collectives of workers who were exposed to air concentrations of up to 0.15 p.p.m. for 8 h and of up to 1.13 p.p.m. for 12 h respectively, the amount of S-PMA in the first urine samples after the shift was significantly higher than in samples collected at the beginning of the shift (P = 0.01). In the first collective the mean values and the standard deviations of the S-PMA concentrations in the samples at the beginning of the shift were 12.0 +/- 16.7 compared with 48.5 +/- 64.5 micrograms/g creatinine at shift end. In the second collective they were 25.1 +/- 25.1 compared with 70.9 +/- 109.2 micrograms/g creatinine. The level of significance of the difference between the concentration values of S-PMA at the beginning and end of the shift was P = 0.01. The phenol concentration did not differ significantly. These results suggest that S-PMA can be regarded as a useful indicator for monitoring individuals and collectives exposed to benzene at levels even less than 1 p.p.m.

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TL;DR: Metabolic studies in animals lead us to believe that MeIQx in the diet is efficiently absorbed and extensively biotransformed.
Abstract: A gas chromatographic-mass spectrometric assay has been developed for the measurement of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in urine. The method employs capillary column gas chromatography, electron capture negative-ion chemical ionization mass spectrometry, a stable isotope-labelled analogue of MeIQx as internal standard and has a limit of detection of 5 pg MeIQx/ml urine. Six subjects consumed a fried beef meal and urine was collected before and after this event. While no MeIQx could be detected in urine collections made prior to meat consumption, the 12-h urine collections of all six subjects made after the meal contained the amine. When the amounts of MeIQx measured in the urine collections were compared to the quantities of amine ingested in the fried beef, it was found that 1.8-4.9% of the oral dose was excreted unchanged in urine. Metabolic studies in animals lead us to believe that MeIQx in the diet is efficiently absorbed and extensively biotransformed.

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TL;DR: In this article, a low SAM/SAH ratio was coupled, in nodules, with a high labeling index (LI), 2-fold fall in 5mC DNA content, increase in c-myc, c-Ha-ras and c-Ki-ras expression and hypomethylation of CCGG sequences in the DNA hybridizing with the three protooncogenes.
Abstract: S-adenosylmethionine:S-adenosylhomocysteine (SAM/SAH) ratio, 5-methylcytosine (5mC) DNA content, and methylation and expression of c-myc, c-Ha-ras and c-Ki-ras have been studied in liver nodules, induced by diethylnitrosamine according to the 'resistant hepatocyte' model, and in regenerating liver (RL) between 0.5 and 72 h after partial hepatectomy (PH). Nodules, 11, 13 and 21 weeks after initiation, grew actively, showed a low tendency to remodel (persistent nodules), and did not exhibit carcinomatous changes. They underwent extensive remodeling after a 1-week SAM treatment (64 mumol/kg/day), and decreased in size and number after a 3-11-week treatment. A low SAM/SAH ratio was coupled, in nodules, with a high labeling index (LI), 2-fold fall in 5mC DNA content, increase in c-myc, c-Ha-ras and c-Ki-ras expression and hypomethylation of CCGG sequences in the DNA hybridizing with the three protooncogenes. In RL a low SAM/SAH ratio, overall DNA hypomethylation and enhanced c-myc expression were first observed 0.5 h after PH, reached a peak at 5 h and progressively returned to pre-PH levels later on. Maximum expression of c-Ha-ras and c-Ki-ras occurred 24-30 h after PH, roughly coincident with the LI peak. However, no great modifications of the methylation pattern of protooncogene CCGG sequence occurred at any time after PH, indicating the presence of hypomethylated genes and/or DNA sequences different from those investigated in this paper. SAM injection to nodule-bearing rats, for 1-11 weeks before killing, and to hepatectomized rats, 2 days before PH and then up to killing, largely prevented decrease in the SAM/SAH ratio and overall DNA methylation and inhibited LI and protooncogene expression. In nodules these effects were proportional to the treatment length and coupled with methylation of CpG residues in the CCGG sequence of the three protooncogenes studied. SAM treatment left the methylation pattern of these genes unchanged in RL. Kinetics of increase in protooncogene expression suggest a role in the regulation of cell cycle in RL. However, decrease in the SAM/SAH ratio, protooncogene hypomethylation and enhanced expression are apparently stable in nodules 11-21 weeks after initiation and could be implicated in continuous nodule growth and progression. Control of DNA methylation and gene expression by exogenous SAM could be a mechanism of the SAM anti-progression effect.

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TL;DR: Dietary additions of each of the monocyclic terpenes, d-limonene or (-)-menthol resulted in a significant inhibition of mammary carcinogenesis and menthol was found to be a more potent chemopreventive agent than limonene during the DMBA initiation of rat mammary tumors.
Abstract: We have previously demonstrated the cancer chemopreventive activities of the monocyclic unsaturated monoterpene d-limonene. In the present work we report data evaluating the chemopreventive effects of limonene and five other monoterpenes with various chemical structures using a 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinogenesis model. The terpenes tested include: oxygenated [(-)-menthol] and non-oxygenated (d-limonene) monocyclic forms, oxygenated (1,8-cineole) and non-oxygenated [(+/-)-alpha-pinene] bicyclic forms and oxygenated [(+/-)-linalool] and non-oxygenated (beta-myrcene) acyclic forms. Dietary additions of each of the monocyclic terpenes, d-limonene or (-)-menthol resulted in a significant inhibition of mammary carcinogenesis. Furthermore, menthol was found to be a more potent chemopreventive agent than limonene during the DMBA initiation of rat mammary tumors. The acyclic and bicyclic terpenes had no significant chemopreventive activities at the dose levels used in these studies.