Showing papers in "Carcinogenesis in 1987"

Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid.

Abstract: Fried ground beef contains substances that inhibit mutagenesis in bacteria and the initiation of epidermal carcinogenesis in mice by 7,12-dimethylbenz [a]anthracene (DMBA). The inhibitors apparently act at least in part via inhibition of cytochrome P-450 activity. A highly purified fraction that inhibited cytochrome P-450 activity in vitro was isolated by HPLC and characterized by GC-MS, and by UV and proton NMR spectroscopy. The fraction contained four isomeric derivatives of linoleic acid each containing a conjugated double-bond system (designated CLA). Synthetically prepared CLA (containing all four isomers) was tested for anti-initiation activity in the two-stage mouse epidermal carcinogenesis system. Seven days, 3 days and 5 min prior to DMBA application, CLA was applied at doses of 20, 20 and 10 mg respectively. Control mice were treated similarly with linoleic acid or solvent (acetone). One week after initiation, and twice weekly thereafter, all mice were treated with 12-O-tetradecanoylphorbol-13-acetate to effect tumor promotion. There was no difference in tumor incidence or yield between linoleic acid-treated mice and solvent-treated control mice. By contrast, the CLA-treated mice developed only about half as many papillomas and exhibited a lower tumor incidence compared with the control mice.

A precursor—product relationship exists between oval cells and hepatocytes in rat liver

Abstract: Oval cell proliferation was induced in twelve male Fischer rats by administration of 2-acetylaminofluorene (2-AAF) for 2 weeks and by performing partial hepatectomy one week after the beginning of 2-AAF administration. Albumin expression in liver was studied by using in situ hybridization of 3H-labeled rat albumin riboprobe. Radiolabeled thymidine was administered to a group of animals at day 6 after partial hepatectomy. The animals were killed at 0, 3, 7, 9, 11 and 13 days after partial hepatectomy. Both oval cells and basophilic hepatocytes showed a prominent expression of albumin, whereas albumin expression in acidophilic pre-existing hepatocytes was decreased. Oval cell nuclei were exclusively labeled one day after administration of [3H]thymidine. At day 9, 11 and 13 basophilic hepatocytes became labeled and the area occupied by these cells increased. This is the first demonstration of the transfer of radiolabeled thymidine from oval cells to newly formed hepatocytes in vivo. Thus the precursor-product relationship between oval cells and basophilic hepatocytes has been established.

Oral administration of the renal carcinogen, potassium bromate, specifically produces 8-hydroxydeoxyguanosine in rat target organ DNA.

Abstract: Following oral administration of a renal carcinogen, potassium bromate (KBrO3), to the rat, a significant increase of 8-hydroxydeoxyguanosine (8-OH-dG) in kidney DNA was observed. In the liver, a non-target tissue, the increase in 8-OH-dG was not significant. The non carcinogenic oxidants, NaCIO and NaCIO2, had no effect on 8-OH-dG formation in kidney DNA. These results suggest that formation of 8-OH-dG in tissue DNA is closely related to KBrO3 carcinogenesis.

Peroxisome proliferation and hepatocarcinogenesis.

Abstract: Peroxisome proliferators constitute a novel class of non-mutagenic hepatocarcinogens, all of which induce a similar pleiotropic response consisting of hepatomegaly, proliferation of peroxisomes in the liver parenchyma] cells and the induction of several hepatic enzymes, particularly those of the peroxisomal fatty acid /3oxidation system (1—3). Presently several structurally dissimilar hypolipidemic compounds, including the widely used drug clofibrate, and certain phthalate ester plasticizers are the two major categories of agents that are recognized as peroxisome proliferators (2). The lack of mutagenicity of these agents led to the proposal that hepatocarcinogenesis is not related to the direct initiating effect of these chemicals (or their possible metabolites), but linked to metabolic disturbance(s) emanating from sustained increase in the number of peroxisomes in liver cells (4). Elucidation of the mechanism of induction of peroxisome proliferation and associated enzymes by these agents is, therefore, considered essential in order to understand the role of peroxisomes in liver carcinogenesis induced by these xenobiotics which do not appear to interact with and damage DNA (5,6). This commentary is a brief review of the biological effects of peroxisome proliferators and of possible mechanisms of induction of pleiotropic responses leading to the development of hepatocellular carcinomas, focusing in particular on the hypothesis that these agents exert their effects by interacting with a specific receptor(s).

Diallyl sulfide, a flavor component of garlic (allium sativum), inhibits dimethyihydrazine-induced colon cancer

Abstract: Diallyl sulfide, a thioether found naturally in garlic, when given by gavage to C57BL/6J mice inhibited by 74% the incidence and reduced the frequency of colorectal adenocarcinoma induced by 20 weekly injections of 1,2-dimethylhydrazine. This result was predicted from a short-term assay measuring defects in nuclear morphology in mouse colon epithelial cells. This chemical is representative of a class of naturally occurring sulfur compounds with profound pharmacologic activity, one aspect of which may be cancer prevention.

Peroxyl free radicals: potential mediators of tumor initiation and promotion

Abstract: In recent years, there has been an explosion of interest in the involvement of free radicals in carcinogenesis (1 -4) . This seems a natural outgrowth of the long overdue realization that free radicals are formed in biological systems and that they contribute significantly to physiological and pathological processes. Much of the emphasis of research linking free radicals and cancer has focused on intermediates of oxygen reduction such as superoxide anion and hydroxyl radical (2). There is another family of oxygencentered free radicals that have received less attention but may be just as important biologically as reduced oxygen intermediates. This Commentary is intended to familiarize the reader with some of the properties of peroxyl radicals and to explain how these species participate in reactions that are relevant to tumor initiation and promotion. Experimental data are presented that are intended to be provocative rather than definitive because this area of investigation is just beginning to evolve.

Isolation and characterization of the major serum albumin adduct formed by aflatoxin B1 in vivo in rats.

Abstract: Aflatoxin B1 (AFB1) was shown to react primarily with one or more lysine residues in serum albumin (SA), accounting for more than half of the total binding to this protein. The radioactivity associated with SA following administration of [U-14C]AFB1 to rats was cleared with a half-life of 2.5 days, which is not significantly different from the half-life of unmodified albumin in the normal rat. The product isolated from a Pronase digest of in vivo-modified SA was identical by chromatographic retention time and u.v. and mass spectroscopy to the synthetic product obtained by the acylase-catalyzed deacetylation of the reaction product of N alpha-acetyl-L-lysine with 8,9-dihydro-8,9-dibromo-AFB1. The latter was characterized by u.v., fluorescence, 500 MHz 1H-n.m.r. and fast atom bombardment mass spectrometry. The spectral data strongly support a structure in which the terminal dihydrofuran ring of AFB1 has been converted to a pyrrolinone ring. It is proposed that the initial adduct is formed by condensation of the dialdehyde tautomer of 8,9-dihydro-8,9-dihydroxy-AFB1, with the epsilon-amino group of lysine, to form a Schiff base, and that the Schiff base undergoes an Amadori rearrangement to an alpha-amino ketone. The pyrrolinone ring is formed by condensation of the amino group with the remaining aldehyde to yield the final product. The purified product was relatively stable but was shown to decompose significantly under the conditions used to isolate it from modified SA.

Bryostatin 1, an activator of protein kinase C, inhibits tumor promotion by phorbol esters in SENCAR mouse skin

Abstract: Bryostatin 1, like the phorbol esters, activates protein kinase C However, bryostatin 1 induces only some of the effects in cultured cells which result from phorbol ester treatment, whereas it blocks other responses to the phorbol esters In mouse keratinocytes in particular, bryostatin 1 induces ornithine decarboxylase, a marker of proliferation, but blocks induction of markers of differentiation Because of the postulated role of induction of differentiation in tumor promotion, we have now examined bryostatin 1 as a tumor promoter and as an inhibitor of phorbol ester tumor promotion in the initiation-promotion model of skin carcinogenesis After initiation with 7,12-dimethylbenz[a]anthracene, weekly topical treatments of the backs of mice with 1 microgram (11 nmol) bryostatin 1 induced epidermal hyperplasia and inflammation, although not to the extent seen after treatment with the promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) Treatment with bryostatin 15 min before each TPA exposure reduced the phorbol ester-induced hyperplasia Bryostatin 1 was ineffective as a complete tumor promoter and displayed very weak activity as a second stage promoter upon treatment of initiated mice for 30 weeks Combined exposure of mice to bryostatin 1 and TPA resulted in a substantial inhibition of promotion by TPA Our in vivo results extend earlier in vitro findings that bryostatin 1 acts as a partial inhibitor of protein kinase C function

Nitromation of amines by stimulated macrophages

Abstract: Rats and mice treated in vivo with Escherichia coli lipopolysaccharide (LPS) synthesize and excrete large quantities of nitrate. Murine peritoneal macrophages, elicited in vivo with thioglycolate and stimulated in vitro with LPS and/or gamma-interferon (IFN), produce copious amounts of nitrate and nitrite. We report here experiments showing N-nitrosamine formation by macrophages immunostimulated in vitro. Macrophage cell lines J774.1, PU5-1.8, WEHI-3 and RAW 264 and freshly isolated macrophages from C3H/He mice were used. Macrophages were cultured in Dulbecco's modified Eagle's medium (pH 7.5) supplemented with calf serum (10%). Supernatant NO2- and NO3- were measured. N-Nitrosamines were extracted with dichloromethane and the extracts analyzed by a gas chromatography--thermal energy analyzer. Cells (1.5 X 10(6)/ml) were incubated with LPS (10 micrograms/ml) and morpholine (15 mM) for 72 h at 37 degrees C. Under these conditions, all of the cell types listed above produced nitrite (40-70 microM) and N-nitrosomorpholine (NMOR; 114-940 nM). LPS was required for both processes, and this effect was enhanced by IFN. Nitrite (150 microM) incubated with morpholine in cell-free medium did not form NMOR nor did cells plus morpholine and NO2-. The rate of NMOR formation in the J774.1 cell line was highest in the middle incubation period (24-36 h) although [NO2-] was highest in the final incubation period (48-72 h). Thus, the cells do not catalyze nitrosamine formation per se, rather the amine traps out a reactive nitrosating species prior to the formation of NO2- and NO3-. These results suggest that immunostimulated macrophages may be capable of nitrosamine formation under physiological conditions.

The effects of low-energy 60-Hz environmental electromagnetic fields upon the growth-related enzyme ornithine decarboxylase.

Abstract: People living in the industrial society of today are unavoidably exposed to low-energy electromagnetic (EM) radiation. The potential risk to human health of such exposure has received much study. In this regard, numerous epidemiological studies have linked exposure to low-energy EM fields to increased cancer risk. We investigated the ability of low-energy 60-Hz EM fields to alter the activity of ornithine decarboxylase (ODC) in a number of established cell lines. The activity of ODC, the controlling enzyme in polyamine biosynthesis, has been shown to be elevated in growing cells or tissues and during the process of tumor promotion. A 1-h exposure to a 60-Hz EM field of an intensity of 10 mV/cm produced a 5-fold increase in ODC activity in human lymphoma CEM cells and a 2- to 3-fold increase in mouse myeloma cells (P3) relative to the unexposed cultures. Depending upon the cell type, ODC activity increased during the 1-h exposure period and remained elevated for several hours after the field exposure ended. In another series of experiments, fields of an intensity as low as 0.1 mV/cm for a 1-h period produced a 30% increase in the activity of ODC in Reuber H35 hepatoma cells grown in monolayer culture. In the H35 cells, continuous exposure to the 60-Hz EM field (10 mV/cm) for periods of 2 and 3 h resulted in either no increase in ODC activity (2 h) or a decrease in enzyme activity (3 h) compared to the unexposed control cultures. The data is discussed in relation to possible molecular mechanisms of field-cell interaction, the importance of the exposure intervals altering cellular ODC activity and the potential ability of 60-Hz EM fields to serve as a tumor promoting stimulus.

A method to quantitate the relative initiating and promoting potencies of hepatocarcinogenic agents in their dose-response relationships to altered hepatic foci.

Abstract: The relative response to various initiating doses of diethylnitrosamine (DEN) and dimethylbenz[a]anthracene of the induction of numbers and size (vol. % of liver) of altered hepatic foci (AHF) in livers of adult female rats of the Sprague-Dawley and Fischer 344 (F-344) strains was studied by methods of quantitative stereology in the presence and absence of the promoting agent, phenobarbital (PB, 0.05% in the diet). In all cases, a relatively linear response with dose, even at the lowest doses employed, was obtained except for the numbers of AHF at the highest dose of DEN (30 mg/kg), which was not significantly different from that at a dose of 10 mg/kg in F-344 female rats. Similar dose-response data were obtained at various doses of two promoting agents effective in hepatocarcinogenesis, PB and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in livers of F-344 female rats following initiation with DEN (10 mg/kg) 24 h post-70% hepatectomy. The response to these agents exhibited threshold levels below which no increase in number or vol. % of liver of AHF was noted in comparison with that in livers of animals not treated with the promoting agents. At several subthreshold doses of both PB and TCDD an inhibition of AHF formation and growth (measured as vol. % of liver) was observed. Based on quantitative stereologic calculations, parameters for the estimation for the relative potency of chemicals as initiating or promoting agents have been established. These are defined as: initiation index = no. of foci induced X liver-1 X [mmol/kg body wt]-1 and promotion index = Vf/Vc X mmol-1 X weeks-1, where Vf is the total volume fraction (%) occupied by AHF in the livers of rats treated with the test agent and Vc is the total volume of AHF in control animals which have only been initiated. These parameters were calculated for a number of agents based on data published in the literature and from those reported herein. Neither parameter varied significantly with the dose of the initiating agent based on the data in this paper. The range of promotion indices extended over more than eight orders of magnitude, whereas that of initiation indices was much less variable. Such parameters may be useful as quantitative estimates of the potency of hepatocarcinogenic agents, such values having potential application to risk estimations.

Possible mycological etiology of oral mucosal cancer: catalytic potential of infecting Candida albicans and other yeasts in production of N-nitrosobenzylmethylamine

Abstract: Yeasts were isolated from 12 cases of oral precancerous lesions (leukoplakia and erythroleukoplakia) by sampling the lesion as well as normal mucosa of each patient, yielding 21 strains of Candida albicans belonging to 15 biotypes, two strains of C. tropicalis, one strain of C. parapsilosis and two strains of Torulopsis glabrata. Biopsies were obtained from the lesions for histologic examination. The catalytic potential of the yeast strains to form N-nitrosobenzylmethylamine (NBMA) from the precursors N-benzylmethylamine and nitrite was assessed at pH 6.8. The NBMA produced was identified and quantitated by h.p.l.c. and confirmed by g.c.-m.s. Nitrosation rates were calculated as total nitrosamine subtracted the chemically produced nitrosamine, and related to number of yeast cells. The yeast strains differed in nitrosation potential (P less than 0.001), ranking from 0 to 1.2 micrograms NBMA/10(6) cells. Candida albicans strains, belonging to the biotypes 051, 147, 151, 153, 157 and 353, which constitute more rarely occurring biotypes, exhibited the highest nitrosation potential. Candida tropicalis, C. parapsilosis and T. glabrata were ranked lower. Strains with high nitrosation potential were generally isolated from lesions with more advanced precancerous changes. The yeast cells were present in the superficial part of the epithelium of the lesions as branching mycelium, and in some cases extending from the mucosal surface to the deeper epithelial cell layers. This might represent a fungal transportation system which could channel precursors in the saliva at the mucosal surface to the deeper part of the epithelium where the produced nitrosamine could be deposited. Thus, further evidence is provided supporting the hypothesis that certain strains of C. albicans and of other yeasts play a causal role in the development of oral cancer, by means of endogenous nitrosamine production.

Inhibitory effects of benzyl isothiocyanate administered shortly before diethylnitrosamine or benzo[a]pyrene on pulmonary and forestomach neoplasia in A/J mice.

Abstract: The inhibitory effects of benzyl isothiocyanate, a naturally-occurring constituent of cruciferous vegetables, on carcinogenesis of the forestomach and lungs of female A/J mice given dimethylnitrosamine (DEN) or benzo[a]pyrene (BP) was investigated. When administered by gavage 15 min prior to p.o. carcinogen challenge, benzyl isothiocyanate almost completely inhibited forestomach tumor formation resulting from DEN but had no effect on pulmonary tumor formation. With BP as the carcinogen, benzyl isothiocyanate had inhibitory effects on carcinogenesis of both the forestomach and lungs, the latter being greater. The implications for epidemiology studies and for chemopreventive strategies of compounds that inhibit when administered shortly before carcinogen challenge are discussed.

Single GST-P positive liver cells--putative initiated hepatocytes.

Abstract: Immunohistochemical investigation of liver tissue 48 h after single doses of the hepatocarcinogens diethylnitrosamine (DEN), dimethylnitrosamine, aflatoxin B1 and methylazoxymethanol acetate revealed the generation of a population of single glutathione S-transferase placental form (GST-P) positive hepatocytes. Yield was carcinogen dose dependent. Although the mixed function enzyme inducers sodium phenobarbital (PB), methylcholanthrene (Mech), polychlorinated biphenyls (PCB) and isosafrole (IS) did not in themselves induce comparable single cell lesions, their application prior to DEN caused significant alteration in the resultant numbers of GST-P positive hepatocytes observed. While PB and IS were associated with decreased yield, Mech and PCB, in contrast, brought about an increase. The results gained from investigation of the effects of 3-aminobenzamide administration and partial hepatectomy carried out at different times after carcinogen treatment also pointed to an 'initiated' character for the lesions and suggest that GST-P may be a useful marker for analyzing factors relevant to the initiation stage of hepatocarcinogenesis. Thus the interplay between carcinogen metabolism, DNA adduct formation and repair, toxicity and proliferation may be assessable in terms of numbers of enzyme-altered solitary hepatocytes.

Toxic and carcinogenic agents in undiluted mainstream smoke and sidestream smoke of different types of cigarettes

Abstract: The mainstream and sidestream smoke of four types of popular US cigarettes were analyzed for toxic and carcinogenic agents. The cigarettes included one without a filter tip, and one filter cigarette each with medium, low and ultra-low smoke yields. The analyses clearly demonstrated that 12 toxic agents determined in this study were significantly reduced in the mainstream smoke of filter cigarettes, as compared with smoke yields from the nonfilter cigarette. In the case of the ultra-low yield cigarette, mainstream smoke emissions were reduced by about 90%. In contrast to this finding, the emissions of the same toxic and carcinogenic components into sidestream smoke of the filter cigarettes were not greatly reduced. Sidestream smoke is the major contributor to environmental tobacco smoke, to which both smokers and non-smokers are exposed. Although the exposure of the smoker to mainstream smoke components is decreased due to proportionally greater consumption of low and ultra-low yield cigarettes, and lower rates of consumption of cigarettes with high smoke yields, the carcinogenic potential of indoor pollutants originating from tobacco products is not diminished.

Carcinogenicity in mice of a mutagenic compound, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) from cooked foods

Abstract: 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), which is a mutagenic compound present in fried beef and beef extracts, was given orally to CDF1 mice at a concentration of 0.06% in the diet for 84 weeks. Liver tumors were induced in 43% of males and 91% of females fed MeIQx. The incidences of liver tumors in mice of both sexes were significantly higher in groups fed MeIQx than in control groups. The incidences of lung tumors in females fed MeIQx and of lymphomas and leukemias in both sexes fed MeIQx were also significantly higher than in the respective controls.

Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation.

Abstract: Cytotoxicity, morphological neoplastic transformation, cellular uptake and metabolic reduction were determined in BALB/3T3 Cl A31-1-1 cells for trivalent arsenic (sodium arsenite, As3+) and for pentavalent arsenic (sodium arsenate, As5+). The levels of cellular uptake of 73As-labelled sodium arsenite and arsenate were dose-dependent and highest in the first hour. At equimolar concentration (3 X 10(-6) M), cellular uptake was 4-fold higher for As3+ than for As5+. Cytotoxicity was higher for As3+ than for As5+, but when correlated to total As cell burden it showed no significant difference for the two forms. Morphological transformation focus assays showed transforming activity for both As3+ and As5+, with relative transformation frequencies also of approximately 4:1. Recovery from the cytosol after exposure for 1-24 h was greater than 90% for either form of absorbed As. Exposure to As3+ yielded 100% as As3+ in cytosol, but exposure to As5+ yielded greater than 70% as As3+, showing a high rate of intracellular metabolic reduction. No methylated metabolites were detected by ion-exchange chromatography. After 24-h incubation in cell-free medium, oxidation of As3+ to As5+ occurred up to 30% of the dose, but incubation in the presence of cells lowered the oxidation level to 4%. As5+ was recovered unchanged from cell-free medium (24-h incubation), but in the presence of the cells it yielded up to 5% as As3+ within 24 h and the cumulative release of As3+ by cells exposed to As5+ was dose-dependent. Glutathione depletion by diethylmaleate inhibited reduction of As5+ to As3+ by these cells up to 25% of controls, showing that As5+ reduction is partly dependent on glutathione. These results suggest that As3+ is the form responsible for the cytotoxic and transforming effects, independently of the valence state of the inorganic arsenic in the culture medium.

Inhibition of hepatocarcinogenic responses to 1,2-dimethylhydrazine by diallyl sulfide, a component of garlic oil

Abstract: The mechanisms by which diallyl sulfide (DAS), a component of garlic oil, inhibits hepatocarcinogenicity of 1,2-dimethylhydrazine (DMH) were examined in male Fischer 344 rats. Rats were subjected to partial hepatectomy to stimulate hepatocellular proliferation required for initiation by DMH (50-200 mg/kg i.p.) given 12 h later. Initiation was assessed by the numbers of foci and nodules of hepatocytes that were positive for gamma-glutamyl transpeptidase (gamma-GT) or glutathione-S-transferase-P (GST-P) after 6 weeks promotion by orotic acid (1% in semi-purified diet). DAS at doses above 50 mg/kg (by gavage) administered 1 h before DMH (50 mg/kg) partially reduced the numbers of gamma-GT and GST-P-positive foci. By comparison, all doses of DAS (25-100 mg/kg) completely prevented liver necrosis by DMH (200 mg/kg). DAS substantially reduced macromolecular binding of [14C]DMH in cultured liver cells, but had no effect on their levels of glutathione-S-transferase, glutathione reductase or glutathione peroxidase at 18 h. These findings suggest that low dosages of DAS which reduce DMH binding appear more likely to inhibit hepatocarcinogenicity by reducing the promoting influences of post-necrotic regeneration than by preventing initiation.

The regulation of protein kinase C by chenodeoxycholate, deoxycholate and several structurally related bile acids

Abstract: There is increasing evidence that the enzyme protein kinase C (PKC) mediates the action of phorbol ester tumor promoters and also the action of certain growth factors. The present studies indicate that the bile acids chenodeoxycholate and deoxycholate inhibit the Ca2+-phosphatidylserine (PS)-dependent activity of PKC in the presence of 1 mM Ca2+, whereas seven structurally related bile acids do not detectably inhibit the enzyme under these conditions. Chenodeoxycholate and deoxycholate appear to inhibit PKC by interactions with both Ca2+ and PS, since their inhibitory potencies are reduced at an elevated PS concentration and since both of these bile acids actually enhance PKC activity, approximately 2-fold, when assayed at an elevated Ca2+ concentration (2 mM). Seven related bile acids also caused an approximately 2-fold enhancement of PKC activity in the presence of 2 mM Ca2+. Chenodeoxycholate and deoxycholate also caused an approximately 1.3-fold enhancement of PKC activity in the presence of 12-O-tetradecanoyl phorbol 13-acetate (TPA) and PS, and the absence of added Ca2+. Thus, depending on the reaction conditions, specific bile acids can act directly to inhibit or enhance PKC activity. There is evidence that during colon cancer formation, both in rodents and in humans, bile acids may act as tumor promoters. Thus the mediation of tumor promotion by bile acids in vivo may involve direct activation of PKC by the bile acids themselves. The present results suggest that the relative extents of absorption of Ca2+ and bile acids by the colonic mucosa may alter the activity of PKC in the mucosa, and thus alter the growth properties of this tissue. The present studies also suggest that lipophilic anionic compounds may provide a new approach to developing therapeutic agents that act by modulating PKC.

Expression of class Pi glutathione transferase in human malignant melanoma cells.

Abstract: The occurrence of glutathione transferase in human malignant melanoma cell lines and solid tumor material has been analyzed and compared with the enzyme composition in fibroblasts and naevus samples. All cells and tissues investigated contained essentially only the acidic class Pi glutathione transferase as demonstrated by SDS-PAGE and immunoblotting. The enzyme was purified from tumor material and characterized. Its intracellular concentration was significantly higher in all the melanoma cell preparations analyzed than in the non-malignant cells, supporting the view that the class Pi glutathione transferase may contribute to the drug resistance that is characteristic of malignant melanoma.

Hydrogen peroxide formation and DNA base modification by tumor promoter-activated polymorphonuclear leukocytes.

Abstract: This report shows that generation of hydrogen peroxide (H2O2) by human polymorphonuclear leukocytes (PMNs) activated with tumor promoters of varying potency as first and second stage promoters correlates well with activities of these promoters in vivo. Those tested were 12-O-tetradecanoylphorbol-13-acetate (TPA), a complete promoter, 12-O-retinoylphorbol-13-acetate (RPA), a synthetic TPA derivative almost devoid of first stage activity in some strains of mice, and mezerein (Mez), a potent second stage and much weaker first stage promoter. Mez-stimulated PMNs produced up to four times less H2O2, whereas RPA-stimulated PMNs produced up to 10 times less H2O2 than TPA-activated cells when used at concentrations between 0.5 and 15 nM to activate 7.5-8.5 X 10(4) PMNs/ml. Phorbol, a non-promoter, was totally inactive in this assay. Furthermore, the tumor promoter-activated PMNs caused formation of 5-hydroxymethyl-2'-deoxyuridine (HMdU) and thymidine glycol (dTG) in DNA co-incubated with those cells. The amounts of modified thymidines formed, particularly of HMdU, correlated well with first stage tumor promoting efficacy and with the amount of H2O2 that was generated by promoter activated PMNs. In comparison with TPA, Mez- or RPA-stimulated PMNs induced formation of 25 or 70% less H2O2 and 30 or 75% less HMdU, respectively, under conditions favoring HMdU formation. Thus, formation of either H2O2 by tumor promoter-stimulated phagocytes or HMdU in DNA exposed to those activated cells may serve as a measure of potency as a first stage tumor promoter. Formation of modified bases such as HMdU in DNA might constitute the genetic change imparted by the first stage tumor promoters.

Bacterially catalysed N -nitrosation reactions and their relative importance in the human stomach

Abstract: Human exposure to endogenously formed N-nitroso compounds has frequently been suggested as a causative factor in carcinogenesis where this is related to chronic bacterial infection such as is seen in gastric achlorhydria. At least two distinct mechanisms of endogenous formation have been identified. The first, a direct chemical reaction between secondary amino compounds and nitrite, is strongly pH dependent and does not proceed rapidly at neutral pH even in the presence of chemical catalysts. The second depends on the direct bacterial catalysis of N-nitrosation. The data presented demonstrate that the bacterially mediated reaction is catalysed by bacterial enzyme systems and proceeds much more rapidly at neutral pH than the chemical reaction. This suggests a particular relevance to the in vivo situation where neutral pH, bacteria and elevated nitrite concentrations are found. Drawing on the kinetic information presented regarding the bacterially mediated nitrosation reaction, the known kinetics of the chemical reaction and the published values for the relevant substrate concentrations in both the colonised and the normal acid stomach the bacterial and chemical reactions have been compared. Using these criteria, and assuming the presence of bacteria with the appropriate metabolic activity, it may be predicted that N-nitroso compounds may be formed in the colonized stomach at much higher concentrations than in the normal acid stomach. The difference in yield may be by two to four orders of magnitude. Different bacterial species and different isolates of the same species show considerable variation in their abilities to catalyse N-nitrosation reactions. The most rapid catalysis is associated with those bacteria capable of reducing nitrate and nitrite by the process of denitrification. The most significant clinical corollary of these studies is that although bacterial catalysis of N-nitrosation has been demonstrated unequivocally, bacterial colonization of the stomach may not itself necessarily result in elevated endogenous N-nitroso compound exposure despite the elevated nitrite concentrations normally associated with such colonization. An increase in exposure to endogenously formed N-nitroso compounds would only be predicted in those individuals where a significant proportion of the colonizing bacteria expressed significant N-nitrosation activity. As a consequence the carcinogenic risk may be restricted to only a small proportion of colonized individuals depending on the prevalence of sustained infection by bacteria with significant N-nitrosation activity, particularly denitrifiers.

Chymotrypsin-specific protease inhibitors decrease H2O2 formation by activated human polymorphonuclear leukocytes

Abstract: Stimulated phagocytic cells generate active oxygen species which are known to contribute to inflammatory diseases, necrosis of surrounding tissues, mutagenicity and carcinogenicity. Until now, it was not certain whether protease inhibitors are capable of decreasing the production of those oxygen species, and if they are, what type of protease inhibitor is the most active. In this work we monitored formation of H2O2 by 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated polymorphonuclear leukocytes (PMNs) because H2O2 is the immediate precursor of the actual damaging species. These determinations were carried out in the absence or presence of protease inhibitors and/or superoxide dismutase (SOD). The protease inhibitors tested were: potato inhibitors 1 (PtI-1) and 2 (PtI-2), a chymotrypsin-inhibitory fragment of PtI-2 (PCI-2), chicken ovoinhibitor (COI), turkey ovomucoid ovoinhibitor (TOOI), Bowman-Birk inhibitor (BBI), lima bean inhibitor (LBI) and soybean (Kunitz) trypsin inhibitor (SBTI). The order of activity, as measured by inhibition of H2O2 formation by TPA-activated PMNs during incubation at 37 degrees C for 30 min, was (in descending order): PtI-1 greater than or equal to PCI-2 greater than PtI-2 greater than COI greater than BBI greater than or equal to TOOI greater than LBI greater than SBTI. Thus, the most effective were the chymotrypsin-specific inhibitors PtI-1 and PCI-2, followed by the bifunctional inhibitors recognizing both chymotrypsin and trypsin, and the least active was SBTI, a predominantly trypsin inhibitor. At the higher concentrations of protease inhibitors tested, the inhibitory activity was similar in both the absence and presence of SOD. These results show that protease inhibitors specific for chymotrypsin but not those that are trypsin-specific are capable of inhibiting formation of active oxygen species during the oxidative burst of stimulated human PMNs.

Quantitative stereological evaluation of four histochemical markers of altered foci in multistage hepatocarcinogenesis in the rat

Abstract: Female F344/N rats dosed with diethylnitrosamine (DEN) 24 h after partial hepatectomy were treated with the promoting agents, phenobarbital (PB) or 3,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or the peroxisome proliferating agent, WY 14,643, for 6 months. Another group was subjected to the Solt-Farber protocol. Altered hepatic foci (AHF) were analyzed by quantitative stereology from frozen serial sections stained for gamma-glutamyl transferase (GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6Pase) and the placental isozyme of glutathione S-transferase (PGST). PGST scored more foci in all groups than GGT and ATPase. PGST marked greater focal volume than GGT or ATPase, and PGST marked focal volume equal to or greater than G6Pase in rats treated with PB, TCDD or the Solt-Farber protocol. However, after treatment with WY 14,643, GGT and PGST marked much less focal volume than ATPase or G6Pase, and PGST scored fewer foci than G6Pase. Numerical estimations of foci scored by those markers on the basis of area of the entire tissue section (per cm2) were relatively different from those values determined by quantitative stereology. While these results confirm earlier studies, they demonstrate the importance of quantitative stereologic analysis of AHF during multistage hepatocarcinogenesis.

Stimulation of DNA synthesis in rat and mouse liver by various tumor promoters

Abstract: In order to investigate whether the stimulation of liver DNA synthesis might be used to detect one class of hepatic tumor promoters, the incorporation of orally administered radiolabelled thymidine into liver DNA was determined in rats and mice 24 h after a single oral gavage of test compounds at various dose levels. Three DNA-binding hepatocarcinogens, aflatoxin B1, benzidine and carbon tetrachloride, did not stimulate but rather inhibited DNA synthesis (not for CCl4). Four hepatic tumor promoters, clofibrate, DDT, phenobarbital and thioacetamide, gave rise to a stimulation in a dose-dependent manner. Single oral doses between 0.02 and 0.3 mmol/kg were required to double the level of thymidine incorporation into liver DNA (= doubling dose, DD). Differences between species or sex as observed in long-term carcinogenicity studies were reflected by a different stimulation of liver DNA synthesis. In agreement with the bioassay data, aldrin was positive only in male mice (DD = 0.007 mmol/kg) but not in male rats of female mice. 2,3,7,8-TCDD was positive in male mice (DD = 10(-6) mmol/kg) and in female rats (DD = 2 X 10(-6) mmol/kg) but not in male rats. The assay was also able to distinguish between structural isomers with different carcinogenicities. [alpha]Hexachlorocyclohexane stimulated liver DNA synthesis with a doubling dose of about 0.2 mmol/kg in male rats whereas the [gamma]-isomer was ineffective even at 1 mmol/kg. So far, only one result was inconsistent with carcinogenicity bioassay data. The different carcinogenicity of di(2-ethylhexyl)adipate (negative in rats) and di(2-ethylhexyl)phthalate (positive) was not detectable. Both plasticizers were positive in this short-term system with DD's of 0.7 mmol/kg for DEHA and 0.5 mmol/kg for DEHP. The proposed assay is discussed as an attempt to devise short-term assays for carcinogens not detected by the routine genotoxicity test systems.

Mutagenicity studies of different polycyclic aromatic hydrocarbons: the significance of enzymatic factors and molecular structure.

Abstract: Dependence of polycyclic aromatic hydrocarbon (PAH)-induced mutagenicity on the bay region of the molecule and on the activating cytochrome P-450 enzyme was studied. Eleven PAHs with and six without a bay region were activated by postmitochondrial supernatants from control and 3-methylcholanthrene (MC)-pretreated C57BL/6 mice and from control, MC- and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-pretreated DBA/2 mice and from control and MC-pretreated Sprague-Dawley and Lewis rats. S-9 fractions from MC- or TCDD-treated animals induced more mutagenicity with PAHs with a bay region compared with S-9 fractions from control animals or MC-treated D2 mice. Mutagenicities of PAHs without a bay region were largely independent of the source of activating enzyme. There were three exceptions, namely benzo[e]pyrene, phenanthrene and perylene (each possessing a bay region), which were not mutagenic. These studies support the notion that the Ah-locus-controlled induction of cytochrome P1-450 activating PAHs into reactive intermediates at the bay region of the hydrocarbon molecule is of prime importance in the mutagenicity of PAHs. Qualitative correspondence to carcinogenicity is also apparent.

Role of cytochrome P1 in the induction of NAD(P)H:quinone reductase in a murine hepatoma cell line and its mutants

Abstract: NAD(P)H:Quinone oxidoreductase (QR) is a widely-distributed enzyme that promotes obligatory two-electron reductions of quinones and thereby protects cells against the cytotoxicity of quinones and their metabolic precursors. QR is induced by a wide variety of chemoprotectors in many animal tissues as well as in the Hepa 1c1c7 murine hepatoma cell line. Such inducers fall into two families: dual inducers (e.g. polycyclic aromatics, azo dyes, beta-naphthoflavone) that elevate QR as well as cytochrome P1-450, and selective inducers of QR (e.g. tert-butylhydroquinone and other redox-labile diphenols). Induction by the first family of inducers depends on binding to the Ah (Aryl hydrocarbon) receptor and the associated expression of a functional cytochrome P1-450 enzyme, whereas the induction by redox-labile diphenols does not appear to be receptor-mediated. In order to analyze the possible role of the cytochrome P1-450 system in the induction of QR, we examined this process in the Hepa 1c1c7 cells and in four mutants of this cell line that are defective in the induction or expression of functional cytochrome P1-450. tert-Butylhydroquinone was an effective inducer of QR in all of the cell lines, and this process does not, therefore, depend on a functional cytochrome P1-450 enzyme. In contrast, azo dyes and polycyclic aromatics induce QR in the parent cell line but not in the various types of cytochrome P1-450-defective mutants. We conclude that the Ah receptor and cytochrome P1-450 function are involved in the induction of QR by certain azo dyes and polycyclic aromatics, but not by phenolic antioxidants.

Prostaglandin H synthase-dependent mutagenic activation of heterocyclic aromatic amines of the IQ-type.

Abstract: Microsomes from ram seminal vesicles known as a rich source of prostaglandin H synthase (PHS) activate the food mutagen IQ (2-amino-3-methylimidazo[4,5-f]quinoline) to (a) product(s) mutagenic in Salmonella typhimurium TA98. The activation is dependent on the PHS cofactor arachidonic acid and is strongly inhibited by the PHS inhibitor indomethacin. In this system, the mutagenic potency of IQ is 22 and 110 times higher than that of 2-aminofluorene and benzidine, respectively. The high mutagenic potency of IQ observed previously with mono-oxygenase activation is thus extended to the PHS system. The mutagenic activity produced by PHS increases for 4 h; this contrasts with the relatively short lifetime of the activity produced by mono-oxygenase and suggests that different agents are involved in the two processes. The PHS-mediated mutagenic activity of IQ is strongly dependent on the bacterial O-acetyltransferase which is defective in strain TA98/1,8-DNP6. Further, the responses of the strains TA1978 and TA1538 indicate that the mutagenic activity is dependent on lack of the bacterial DNA excision repair and independent of the plasmid pkM101 coded error-prone DNA repair system. Structural analogs of IQ without a methyl group on the imidazole ring and with a naphthalene instead of the quinoline ring show greatly diminished PHS-mediated mutagenic activity. The strain response pattern and structure-activity relationships are similar to those found with mono-oxygenase activation of IQ and thus indicate a basic similarity of the IQ activation via PHS with that via mono-oxygenase. It is hypothesized that PHS may activate carcinogenic heterocyclic aromatic amines in vivo.

Influence of ascorbic acid dose on N-nitrosoproline formation in humans.

Abstract: A relationship between ascorbic acid intake and N-nitrosoproline (NPRO) excretion in humans on a controlled diet was established. Seven healthy males were placed on a low nitrate, low ascorbic acid diet for 12 consecutive days. On days 3-12, a 5.24 mmol oral dose of sodium nitrate was administered in mid-afternoon, at least 2 h after the subject's last meal. On days 4-12, a 4.35 mmol oral dose of L-proline was administered 30 min after the nitrate dose. Ascorbic acid was given in amounts which increased daily from day 5 to day 10 (0.01-5.68 mmol; 1.76-1000 mg) with the proline. Total 24 h urines were assayed for nitrate, NPRO and total ascorbic acid. Nitrate balance was monitored using [15N]nitrate. Average endogenous nitrate synthesis was 1.28 +/- 0.43 mmol/day/person. NPRO excretion was reduced by 6 nmol/day when 0.05 mmol of ascorbic acid was administered. However, as much as 5.68 mmol ascorbic acid did not return NPRO excretion to levels observed before the nitrate and proline were administered. More than 10 times the ascorbic acid required to completely inhibit NPRO formation in vitro did not return NPRO excretion to baseline levels. These data indicate that endogenous nitrosation may be more facile than predicted by the in vitro chemistry.

Assay of 1-nitropropane, 2-nitropropane, 1-azoxypropane and 2-azoxypropane for carcinogenicity by gavage in Sprague-Dawley rats.

Abstract: 1-Nitropropane (1-NP), 2-nitropropane (2-NP), 1-azoxypropane (1-AP) and 2-azoxypropane (2-AP), were assayed for carcinogenicity by gavage in male Sprague-Dawley rats. 2-NP was given at 1 mmol/kg three times per week for 16 weeks. 1-NP (1 mmol/kg), 1-AP (0.1 mmol/kg), 2-AP (0.1 mmol/kg) or Emulphor EL-620 vehicle was given three times per week for 16 weeks, and then once per week for 10 weeks. In the 2-NP treated group both benign and malignant liver tumors occurred in 100% of the animals. No treatment related tumors occurred in rats receiving 1-NP. In rats treated with 1-AP, a high incidence of skin tumors (100%), mostly keratoacanthomas, and of tumors of the nasal cavity (59%) was observed. Rats which were given 2-AP also showed an increased incidence of skin keratoacanthomas (21%), but not of other tumors. These findings (i) confirm, using the oral route of administration, the results of inhalation studies by others indicating the potent hepatocarcinogenicity of 2-NP, (ii) establish a practical model in which the mechanism of 2-NP carcinogenicity can now be more readily studied, and (iii) demonstrate that 1-AP, and probably 2-AP, like other aliphatic azoxy compounds thus far examined, are carcinogenic in rats.