Microsome-mediated transacetylation and binding of N-hydroxy-4-aminobiphenyl to nucleic acids by hepatic and bladder tissues from dog. Microsome-mediated metabolism of [3H]4-aminobiphenyl (ABP) and binding of [3H]N-hydroxy-4-aminobiphenyl (N-OH-ABP) to nucleic acids by dog hepatic and bladder microsomes were investigated. HPLC analysis of the ethyl acetate extracts of hepatic microsomal incubates of [3H]ABP in the presence of 4-acetylaminobiphenyl (AABP), N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), or acetyl coenzyme A (AcCoA) as acetyl donors showed the formation of [3H]AABP, suggesting that microsomes catalyze N-acetylation of ABP involving transacetylation. Dog hepatic microsomes also catalyzed the binding of [3H]N-OH-ABP to RNA in the presence of AABP, N-OH-AABP or AcCoA, and the binding was blocked by paraoxon, an inhibitor of microsomal deacetylases. Binding of [3H]N-OH-ABP to DNA was catalyzed also by dog hepatic microsomes, and the extent of binding was 266, 156 and 135 pmol/mg DNA for AABP, N-OH-AABP and AcCoA as acetyl donors respectively. HPLC analyses of the DNA hydrolysates showed that the major adduct formed was N-(deoxyguanosine-8-yl)-4-aminobiphenyl, based on mobility of the adduct in comparison with the synthetic standard. The acetyl adduct N-(deoxyguanosine-8-yl)-4-acetylaminobiphenyl was not detected in the DNA hydrolysates. Adduct profiles obtained from 32P-postlabeling of DNA samples from the microsome-mediated binding of [3H]N-OH-ABP showed similarities to the profile obtained previously from the chemical interaction of N-OH-ABP with DNA under acidic conditions, suggesting that the microsome-mediated binding of N-OH-ABP may proceed via formation of aryl nitrenium ions as the ultimate electrophilic species. Microsomes from dog bladder also catalyzed the binding of [3H]N-OH-ABP to RNA and DNA in the presence of AABP, N-OH-AABP or AcCoA as acetyl donors, though the levels of binding were less than those observed with hepatic microsomes. The prevalence of these acetyl transferases in the target organs for ABP and AABP carcinogenesis raises the possibility that metabolic activation of the proximate metabolite N-OH-ABP could occur directly in these tissues and these reactions could play a critical role in the initiation of cancers.


The role of the human acetylation polymorphism in the metabolic activation of the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The metabolic activation of the heterocyclic food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) by two human cytochrome P450 monoxygenases (P4501A1 and P4501A2) and two human N-acetyltransferases (NAT1 and NAT2) was investigated. Various combinations of these enzymes were functionally expressed in COS-1 cells. DNA adducts resulting from the activation of IQ were assayed quantitatively by the 32P-postlabeling procedure. The highest adduct frequency was observed in cells expressing both CYP1A2 and NAT2. CYP1A2 in combination with NAT1 was 3-6 times less active. When expressed alone these enzymes gave rise to low adduct frequencies. Experiments with N-acetyl-IQ as substrate suggest that NAT1 and NAT2 in addition to their known role in N-acetylation display arylhydroxamic acid N, O-acetyltransferase (AHAT) activity. Quantitative differences in adduct formation between IQ and N-acetyl-IQ indicated that metabolic activation of these arylamines preferentially occurs by P4501A2-catalyzed N-hydroxylation followed by O-acetylation mediated through NAT1 and/or NAT2. These data, in combination with the known genetic polymorphism of NAT2, may explain the clinical observation that the acetylation polymorphism constitutes a risk factor in the carcinogenic activation of environmental mutagens.


DNA adducts and induction of sister chromatid exchanges in the rat following benzo[b]fluoranthene administration. Benzo[b]fluoranthene (B[b]F) was administered (100 mg/kg by i.p. injection) to male Sprague--Dawley rats. Lungs, livers and peripheral blood lymphocytes (PBLs) were harvested 1, 3, 5, 7, 14, 28 and 56 days after treatment. Several DNA adducts were observed in each tissue, with maximal levels occurring at approximately 7 days after treatment. Lung DNA exhibited consistently higher adduct levels than liver or PBL DNA. At 56 days after B[b]F administration, the adducts in liver and PBL DNA were present at < 10 amol/microgram DNA, while in lung there were 100 amoles/microgram DNA. No significant differences were observed between tissues in the types of adducts produced. Co-chromatography with synthetic standards showed that only a minor adduct produced in vivo is derived from trans-9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene-11,12-oxide. Sister chromatid exchanges (SCEs) from whole blood cultures were significantly increased relative to concurrent controls between 1 and 14 days after B[b]F administration, with maximum levels at 14 days. By 28 days after treatment, SCEs had essentially returned to control levels. SCE induction did not correlate with the amount of B[b]F--DNA adducts remaining in the PBLs at harvest time.


Effect of varying the type of fat in a semi-purified AIN-76A diet on cellular proliferation in the mammary gland and intestinal crypts in female Swiss Webster mice. Young virgin female Swiss Webster mice were fed AIN-76A semi-purified diets containing equal weights of different fats for approximately 30 days. Using [3H]thymidine radioautography, it was established that mice fed 100% lard or high levels of fish oils (menhaden oil or cod liver oil) developed elevated cellular proliferation in the duct cells of the mammary gland and an increased number of labeled cells/crypt in the crypts of the colo-rectum accompanied by an increase in the size of the proliferative compartment. A possible inverse correlation between the level of [3H]thymidine labeling in the mammary gland, but not in the colo-rectum, and the linoleic acid content of individual diets may help to explain the significance of these observations. The effect of adding an antioxidant mixture to these diets was to reduce the excess proliferation induced in the intestinal crypts by lard or fish oil to the level induced by soybean oil, but only partially so in the duct cells of the mammary gland.


Depletion of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine in three-dimensional collagen cultures of normal human breast epithelial cells. O6-Alkylguanine--DNA alkyltransferase (AGT) is a protein which removes the promutagenic O6-alkylguanine lesion induced in DNA by alkylating agents. Our results demonstrate that freshly isolated organoids from reduction mammoplasty specimens contain significant levels of AGT activity. AGT activity in breast epithelial cells shows interindividual variation. Constitutive levels of AGT activity remain unchanged during short-term serum-free culture of breast epithelial cells inside three-dimensional rat-tail collagen gel matrix. In the present study, we optimized conditions for depleting AGT activity in human breast epithelial cells cultured in three-dimensional collagen gel matrix using O6-methylguanine and O6-benzylguanine which are substrates for AGT. AGT activity was efficiently inactivated by exposure of cells to O6-methylguanine or O6-benzylguanine. Inactivation with O6-benzylguanine was more rapid, of greater magnitude and consistency and occurred at lower concentrations than with O6-methylguanine. Near-complete inactivation (> 99.5%) of AGT activity was reproducibly achieved with 50 microM O6-benzylguanine. In contrast, 500 microM O6-methylguanine was needed to obtain a maximal effect and this reduced AGT activity by only 53-93% of control. Within 30 min of adding the free base, 50 microM O6-benzylguanine depleted 95% of the levels of AGT compared to 30% inhibition with 500 microM O6-methylguanine. The profile for restoration of AGT activity was different following a 24 h incubation and subsequent removal of each of the guanine derivatives. AGT activity levels remained undetectable for at least 2 days after removal of 50 microM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. AGT activity levels remained undetectable for at least 2 days after removal of 50 microM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. In contrast, following removal of 500 microM O6-methylguanine, the activity was restored from its nadir of 16% of control values reaching pretreatment levels after 5 days. These results suggest that treatment with O6-benzylguanine may be used to modulate the incidence of transforming mutations in cultured human breast epithelial cells treated with chemical carcinogens which give rise to O6-alkylguanine adducts.


Peroxisome proliferation and resistance to hydrogen peroxide in rat hepatocytes: is development of resistance an adaptation to cytotoxicity? In this work the resistance of peroxisome-proliferated hepatocytes to hydrogen peroxide (H2O2) has been studied. The question has been raised as to whether this resistance is a response to cytotoxicity. In an initial series of experiments, hepatocytes were isolated from rats that had been treated with nafenopin (NAF-hepatocytes). Isolated cells were exposed to a H2O2-generating system or to H2O2 in pulses. The ability to attach to collagen was used as a toxicological endpoint. Loss of attachment was found to be correlated to glutathione (GSH) depletion, and NAF-hepatocytes were more resistant to GSH depletion and to loss of attachment induced by H2O2 than were control hepatocytes. NAF-hepatocytes were not resistant to hydroquinone or to adriamycin. It was also indicated that this resistance was related to an altered metabolism of H2O2, less dependent on GSH. In a second series of experiments, hepatocytes from altered hepatic foci-bearing rats, treated with nafenopin or di(2-ethylhexyl)phthalate (DEHP), were used. This model was used in an attempt to monitor the development of resistance in different subpopulations of hepatocytes. It was found that the majority of hepatocytes developed resistance towards H2O2, and that, for example, foci marker-positive hepatocytes were as resistant as marker-negative cells. In control experiments with this model, it was found that marker-positive cells were more resistant towards diethyl maleate (DEM) or phorone than were marker-negative cells. In addition to demonstrating the validity of the model, these control experiments indicate an increased steady-state level of H2O2 in cells from peroxisome proliferator-treated rats. Other control experiments suggested that a low GSH-peroxidase activity protected from, rather than aggravated, the effect of peroxisome proliferation on marker-negative and GSH-depleted cells. It is concluded that H2O2 metabolism may affect the function of collagen receptors, but that a shift in H2O2 metabolism, so that it becomes less dependent on GSH, conferred resistance to this effect. The apparent non-focal induction of resistance to peroxisome proliferators, as opposed to the focal induction of resistance induced by most liver carcinogens, may explain the lack of development of gamma-glutamyltranspeptidase-positive foci in peroxisome proliferator-treated rats.


Analysis of residual amino acid--DNA crosslinks induced in intact cells by nickel and chromium compounds. Chinese hamster ovary cells were incubated with radioactive amino acids, the DNA was isolated by standard proteinase K/phenol/chloroform extraction and residual amino acids complexed to the DNA were examined as an index of metal induced DNA-protein crosslinks. Using this method, both chromate and nickel caused residual histidine and cysteine to be complexed with the DNA isolated from metal-treated cells. In the case of chromate, a number of amino acids were studied and Tyr, Thr and Cys were found to be complexed to DNA at a level (above the untreated control) that was statistically significant. Stability studies indicated that some of the chromate-induced DNA-protein complexes were mediated by direct participation of chromium(III), whereas others that were resistant to dissociation by EDTA and mercaptoethanol did not seem to involve direct chromium(III) participation. A significant portion of the cysteine complexed to DNA by chromate was believed to involve glutathione since treatment of cells with cycloheximide did not decrease chromate-induced cysteine-DNA crosslinks. In the case of nickel, most of the stable DNA-protein crosslinks did not involve direct metal participation and were probably oxidatively mediated by Ni(II)/Ni(III) redox cycling. These findings present new methodology for analysis of DNA-protein crosslinks by examination of residual amino acids associated with the DNA. This method should be highly sensitive and will yield important information about the mechanism of metal-induced DNA-protein crosslinks.


Expression of O6-methylguanine--DNA methyltransferase and uracil--DNA glycosylase in human placentae from smokers and non-smokers. DNA repair capacity is likely to be a critical factor in mutagenesis and carcinogenesis, as well as for the response to some cytostatics. We have studied inter- and intra-individual variation in the activities of O6-methylguanine--DNA methyltransferase (O6-MT) and uracil--DNA glycosylase (UDG) in 35 placentae from smokers and non-smokers. The maximum interindividual variation in the activities of O6-MT and UDG were 8.3- and 7.7-fold, respectively. The corresponding intraindividual variations were 2.7- and 3.3-fold. Generally, a high level of O6-MT activity was accompanied by a high O6-MT mRNA level, but no such correlation was seen for UDG. These results were not due to degradation of the enzymes or mRNAs after delivery. No correlation between the activities of O6-MT and UDG was observed, indicating that they are differentially regulated. A 1.4-fold (P < or = 0.05) higher activity of O6-MT was observed in smokers as compared to non-smokers, indicating a small, but statistically significant difference. No significant difference was observed for UDG. Our results demonstrate that DNA repair capacities vary largely between different individuals, and that environmental factors may modulate the expression of DNA repair enzymes.


11-Methylbenzo[a]pyrene: bay region distortions. Substitution of a methyl group in the 11-position of benzo[a]pyrene (B[a]P) enhances its carcinogenicity. An X-ray crystallographic determination of the three-dimensional structure of 11-methylbenzo[a]pyrene (11-MeB[a]P) shows that steric overcrowding in the bay region is relieved somewhat by distortions of the bay-region bond angles in the plane of the ring system. A comparison with the structure of 7,12-dimethylbenz[a]anthracene (DMBA), which shows out-of-plane distortions to relieve such strain, shows that, in general, H...H intramolecular interactions between neighboring rings in a polycyclic aromatic hydrocarbon are the primary determinants of the nature of the molecular distortions as a result of steric overcrowding (mainly in-plane for 11-MeB[a]P and mainly out-of-plane for DMBA). The 11-MeB[a]P molecule exhibits considerable flexibility as evidenced by slightly different conformations in the two molecules found in the asymmetric unit of the crystal. One molecule is fairly flat with bond angle distortions in the bay region, while the other is slightly buckled as a result of some twist (15 degrees) in the bay region. Computer modeling indicates that steric overcrowding as a result of the bay-region 11-methyl group may affect the conformation of the ring that bears the diol and epoxide groups in the anti-diolepoxide. The nature of this distortion may, in turn, provide a clue to the reason for the greater carcinogenicity of B[a]P when methylated at the 11-position in the non-benzo bay region site. In addition, the 11-methyl group, because of its bulk, may affect the orientation of the polycyclic hydrocarbon as it lies between the nucleic-acid bases when covalently bound to DNA.


Copper-dependent site-specific mutagenesis by benzoyl peroxide in the supF gene of the mutation reporter plasmid pS189. Benzoyl peroxide (BzPO) enhances tumor promotion and malignant conversion in mouse epidermis. DNA damage may contribute to these processes. BzPO reacts with Cu(I) to produce the benzoyloxyl radical, which in turn causes strand breaks in plasmid DNA. In this study we investigated whether BzPO with or without Cu(I) caused promutagenic DNA damage in the supF gene of the mutation reporter plasmid pS189 replicating in human Ad293 cells. Exposure of pS189 in vitro to BzPO (0.1-1 mM) inhibited plasmid replication; however, addition of Cu(I) (0.1 mM) did not augment BzPO-induced plasmid toxicity. Exposure to BzPO with or without 0.1 mM Cu(I) was also associated with a concentration-dependent increase in mutation frequency, up to > 100-fold above the spontaneous mutation frequency. Supplemental Cu(I) was not required for mutagenesis; however, it both raised the maximal mutation frequency observed and lowered the threshold concentration of BzPO necessary to discern mutagenesis above the spontaneous background. Neither the hydroxyl radical scavengers mannitol or DMSO, the spin trap N-tert-butyl-alpha-phenylnitrone, nor reduced glutathione altered BzPO/Cu(I)-induced mutagenesis; however, mutagenesis was suppressed by the chelator EDTA. Twenty-four of 32 individual BzPO/Cu(I)-induced mutants characterized by sequencing contained point mutations; 22/25 point mutations occurred at G-C base pairs. There were five large deletions and four small deletions. Three additional BzPO-induced mutants contained four point mutations, all occurring at G-C base pairs. Two BzPO/Cu(I)-induced mutational clusters at d(pGGG)-d(pCCC) sites were observed. These data suggest that BzPO may interact with Cu(I) bound to G-C base pairs in DNA to produce site-specific promutagenic DNA damage.


Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes. An acetyltransferase-overexpressing strain of Salmonella typhimurium (NM2009) has been used to investigate roles of human liver microsomal cytochrome P450 (P450) enzymes in the activation of carcinogenic nitrosamine derivatives, including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines, to genotoxic products. Studies employing correlation of activities with several P450-dependent monooxygenase reactions in different human liver samples, inhibition of microsomal activities by antibodies raised against human P450 enzymes and by specific P450 inhibitors, and reconstitution of activities with purified P450 enzymes suggest that the tobacco-smoke-related nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N-nitrosonornicotine (NNN) as well as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are oxidized to genotoxic products by different P450 enzymes, particularly P450 2E1 and 2A6. The activation of NDMA and NNN by liver microsomes was suggested to be catalyzed more actively by P450 2E1 than by other P450 enzymes because the activities were well correlated with NDMA N-demethylation and aniline p-hydroxylation in different human samples, and purified P450 2E1 had the highest activities in reconstituted monooxygenase systems. The relatively high contribution of P450 2A6 to the activation of NDEA and NNK was supported by the correlation seen with coumarin 7-hydroxylation in human liver microsomes, and antibodies raised against P450 2A6 inhibited both activities by approximately 50%. P450 3A4, 2D6 and 2C enzymes appear not to be extensively involved in the activation of these nitrosamines as judged by several criteria examined. Thus, this work indicates that several P450 enzymes, particularly P450 2E1 and 2A6, catalyze metabolic activation of nitrosamine derivatives including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines in human liver microsomes.


Identification and quantitative detection of isomeric benzo[a]pyrene diolepoxide--DNA adducts by low-temperature conventional fluorescence methods. The pyrene-like fluorescence of adducts derived from the covalent binding of (+/-)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [+/-]-anti-BPDE] to DNA increases in intensity by factors of 20 or more as the temperature is lowered from ambient to approximately 100 K. This effect is primarily associated with the strong quenching of the pyrene-like fluorescence of BPDE-deoxyguanosyl adducts at room temperature, and the suppression of the electron-transfer quenching mechanism at 100 K. In contrast, the fluorescence of BPDE-deoxyadenosyl adducts is not quenched at ambient temperatures, and the fluorescence yields of (+/-)-anti-BPDE-poly(dA-dT).(dA-dT) adducts increases by only a factor of 2 in this same temperature range. Utilizing an internal fluorescein fluorescence standard to correct for differences in light scattering and variations in instrumental factors, a fluorescence method is described for quantitatively determining the levels of benzo[a]pyrene diolepoxide derivatives covalently bound to cellular DNA at 100 K. The method is illustrated with (+/-)-reverse-BPDE [(+/-)-trans-9,10-dihydroxy-anti-7, 8-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene]. Adduct levels as low as 10 pmol in a 400 microliters sample volume can be detected and identified from their excitation and fluorescence emission spectra using a conventional and commercially available fluorometer. In the case of modified DNA extracted from BPDE-treated Chinese hamster ovary cells or from mouse skin (approximately 1 BPDE residue/20,000 bases), such an analysis requires only 100 micrograms of DNA. Conformationally different adducts derived from the binding of the isomeric (+/-)-anti-BPDE, (+/-)-reverse-BPDE or (+/-)-syn-BPDE to cellular DNA can be distinguished by their low-temperature fluorescence excitation spectra. Specifically, the quasi-intercalated site I BPDE adducts (believed to be associated with cis-addition stereochemistry) can be distinguished from site II adducts situated at external BPDE binding sites (trans-addition stereochemistry). These results suggest that the fates of these conformationally different BPDE-DNA adducts, e.g. due to enzymatic repair, can be monitored as a function of time in DNA extracted from intact, functioning cells.


Influence of caffeic acid and other o-dihydroxybenzene derivatives on N-methyl-N'-nitro-N-nitrosoguanidine-initiated rat forestomach carcinogenesis. Promotion effects of the o-dihydroxybenzene derivatives, protocatechuic acid (PCA), dopamine hydrochloride (DAH), dl-dopa and caffeic acid on forestomach and glandular stomach carcinogenesis were investigated in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Groups of 20 male F344 rats were given a single intragastric administration of 150 mg/kg body wt MNNG and starting 1 week later than received diet containing 1.5% PCA, 1.5% DAH, 1.5% dl-dopa or 1% caffeic acid or basal diet alone for 51 weeks and then killed. Other groups of 10-15 rats were given PCA, DAH, dl-dopa or basal diet alone without the MNNG pretreatment. On histological assessment, the incidences of forestomach papillomas and squamous cell carcinomas were significantly enhanced in the group treated with caffeic acid (95 and 100%) as compared with the control values (35 and 10%). Although the incidence was not different, the number of papillomas per rat in the group given DAH (0.79 +/- 0.79) was also significantly increased (0.35 +/- 0.49). PCA and dl-dopa treatments did not modify the development of neoplastic lesions in the forestomach epithelium to any significant extent. None of the four chemicals enhanced glandular stomach carcinogenesis. The results thus demonstrated that whereas caffeic acid and DAH respectively, exert strong and weak promotion activity for rat forestomach carcinogenesis this promotion potential is not shared by all dihydroxybenzene derivatives. An influence of substituents in the para position in addition to the o-dihydroxy moiety is indicated.


Detection and quantitation of dG-AAI and dA-AAI adducts by 32P-postlabeling methods in urothelium and exfoliated cells in urine of rats treated with aristolochic acid I. Analysis of aristolochic acid I (AAI)-DNA adducts in exfoliated cells in urine, urothelium and entire urinary bladder were studied after oral administration of five daily doses (10 mg/kg body wt) AAI for 3 months to rats. The two major adducts excreted in urine are presumably identical to the two main adducts formed in vitro and in vivo in different organs in the rat, which have previously been characterized in vitro as 7(-deoxyguanosin-N2-yl)-aristolactam I and 7(-deoxyadenosin-N6-yl)-aristolactam I. Urine samples were collected on dry-ice, subsequently pooled and purified according to the protocol of Kadlubar and co-workers. DNA was isolated, digested and AAI-DNA adducts of exfoliated cells in urine and urothelium of rats were detected and quantitated by enhancement methods of the 32P-postlabeling assay, namely nuclease P1 enrichment or butanol extraction. Autoradiograms indicated that adduct patterns in DNA derived from exfoliated cells in urine were very similar to those obtained from DNA isolated from tissues. Quantitative analysis of adducts revealed adduct levels declining for both adducts from DNA isolated from urothelium to DNA isolated from the entire urinary bladder to DNA isolated from exfoliated cells in urine. In general, count rates of two predominant AAI adducts were enhanced by butanol extraction approximately 3- to 8-fold when compared with the nuclease P1 digestion technique. The identity of the two major adducts was confirmed by co-chromatography with eluted spots from in vivo adducts by comparing mobilities on poly-(ethyleneimine)-cellulose plates. Microbiological investigations of the urine revealed no gross contamination with bacteria, so that the isolated DNA supposedly originated from exfoliated urothelial cells. This study indicates that 32P-postlabeling analysis can be used to monitor non-invasively the formation of carcinogen-DNA adducts in animals or humans exposed to carcinogens.


Inhibition of 7,12-dimethylbenz[a]anthracene-induced mammary tumors and DNA adducts by garlic powder. The present studies determined the influence of dietary supplements of garlic powder (0, 1, 2 or 4%) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors and on the in vivo occurrence of mammary DMBA-DNA adducts in rats. Diets were offered 2 weeks before and 2 weeks following DMBA treatment (25 mg/kg body wt). An additional group was fed the 2% garlic powder diet throughout the 20 week study. Although food intake and weight gain were not influenced, dietary garlic powder supplementation did significantly delay the onset of first tumors (P < 0.01) and did reduce the final mammary tumor incidence (P < 0.01). Consumption of garlic powder also significantly depressed the in vivo binding of DMBA to mammary cell DNA. Binding of both anti- and syn-dihydrodiol epoxides to DNA was depressed in rats fed supplemental garlic powder. The activity of glutathione S-transferase (GST) in mammary and liver tissue from rats fed 2% dietary garlic powder was higher than observed in tissues from rats fed the basal diet. No further increase in GST activity occurred when the dietary garlic content was increased from 2 to 4%. Final mammary tumor incidence was found to correlate positively with total DMBA-DNA binding and the quantities of individual DMBA-DNA adducts. The present studies demonstrate that garlic powder is effective in inhibiting DMBA-induced mammary tumors, possibly by reducing DMBA-DNA binding.


Effect of dietary protein level on aflatoxin B1 actions in the liver of weanling rats. The hepatocarcinogenic responses of rats to aflatoxin B1 (AFB1) are believed to depend on microsomal activation of the toxin, followed by macromolecular binding. Dietary protein insufficiency is reported to reduce the level of microsomal metabolism, and therefore would be expected to reduce the AFB1-induced carcinogenicity. Indeed, diminished hepatocarcinogenicity in low-protein diet fed weanling rats that had received AFB1 has been reported. In the present study, carcinogenicity and other toxic effects of AFB1 (0.5 p.p.m.) fed to weanling male Fischer F344 rats on a low-protein diet (5%) or normal-protein (20%) diet for up to 8 weeks were examined. In our study, in contrast with the previous report, all animals that had survived some initial toxicity were found to have developed hepatic tumors or hyperplastic gamma-glutamyltransferase-positive foci a year later. The low-protein diet also produced sub-acute toxicity after AFB1 exposure in the weanling rats, leading to severe histological changes, and the death of about half the animals after 3-4 weeks of exposure. Animals fed an AFB1-containing normal-protein diet also exhibited AFB1-induced hepatocarcinogenicity, but not the sub-acute toxicity. The levels of hepatic enzymes involved in AFB1 metabolism were examined in animals fed the low- or normal-protein diets in the absence of AFB1. The low-protein diet, fed to 3 week weanlings for the subsequent 5 weeks, decreased hepatic cytochrome P450 levels, as well as the in vitro capacity of microsomal fractions to form AFB1-8,9-dihydrodiol, an index of AFB1-8,9-epoxide formation. Rats on a normal-protein diet did not show these changes. This discrepancy between the observed increase in sub-acute toxicity and decrease in microsomal activities in the low-protein fed animals implies that the toxic effects observed in these rats were not directly related to metabolic activation of the toxin. In contrast to the diminished microsomal in vitro AFB1 activation, however, in vivo AFB1-DNA adduct formation ability in rats receiving the low-protein diet in the absence of AFB1 was found to become elevated more rapidly during the 5 week experimental feeding period, compared with animals receiving the normal-protein diet. This was accompanied by a more rapid fall in the levels of AFB1-glutathione S-transferase isozyme activity in the low-protein fed animals. The results of this study on weanling rats support the importance of AFB1-GSH in protecting against the carcinogenic responses to AFB1, and probably also the sub-acute toxicity of the latter.(ABSTRACT TRUNCATED AT 400 WORDS)


Carba-prostacyclin inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation in sensitive murine epidermal JB6 cells. The ability of carba-prostacyclin (cPGI2), a stable analog of prostacyclin (PGI2), to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 cells was investigated. JB6 cells sensitive (P+) and resistant (P-) to TPA-induced transformation to anchorage-independent growth were plated in soft agar in the presence or absence of cPGI2 for 14-21 days. Transformation frequencies were determined by recording colony numbers. cPGI2 was found to inhibit TPA-induced transformation of P+ cells in a dose-dependent fashion with 1 microM cPGI2 producing approximately 50% inhibition of colonies in soft agar. Our findings are consistent with the hypothesis that TPA-induced transformation in JB6 cell variants is mediated by PGI2 via regulation of adenylate cyclase activity and cAMP accumulation, with resultant inhibition of expression of the transformed phenotype, reflected in anchorage-independent growth.


Expression of ras oncogene mRNA and protein in aberrant crypt foci. The expression of the ras oncogene in aberrant crypt foci was studied by both in situ hybridization and immunohistochemical approaches. Aberrant crypt foci are hypothesized to represent the earliest identifiable microscopic lesions of colon cancer in rodent colons. Sprague-Dawley male rats were injected with azoxymethane (20 mg/kg s.c.) once. Twelve weeks later, aberrant crypt foci were identified topographically, microdissected and processed for histology. In situ hybridization with an antisense oligomer of c-ras demonstrated increased expression of ras-specific RNA in aberrant crypts compared to normal crypts. A low amount of non-specific hybridization was obtained with the corresponding sense oligomer. The percentage of cells with grains (labeling index) was calculated in early and advanced aberrant crypt foci. This index was also calculated in normal appearing crypts. The labeling indices for the early and advanced aberrant crypt foci were significantly greater than that of normal crypts (18.0 and 25.0 versus 11.9). In the same tissue specimens, immunohistochemical staining for ras p21 with the monoclonal antibody (Y13-259) revealed strong staining intensity in early aberrant crypts (15/22) and advanced aberrant crypts (22/30) compared to normal crypts (3/50). The immunohistochemical results demonstrate the presence of elevated levels of ras p21 in the same tissue as increased levels of ras-specific message. This investigation provides the earliest demonstration of increased expression of the ras oncogene in precursor lesions of colon cancer possessing dysplastic features.


Adaptation and selection as factors in the spontaneous transformation of NIH-3T3 cells. The effect of calf serum (CS) concentration on the spontaneous transformation of NIH-3T3 cells was investigated. Maintenance of NIH-3T3 cell populations in media containing a low concentration of calf serum (2% CS) increased the proportion of spontaneously transformed cells appearing within a population. Higher CS concentration (10% CS) decreased the proportion of transformed cells in the population. In searching for the reasons for the effect of serum concentration on transformation frequency, three conclusions were reached. (i) Non-focus-forming NIH-3T3 cells exhibit heterogeneity in progression toward transformation. (ii) Low serum concentration can increase the frequency of focus-forming cells within an NIH-3T3 population by decreasing the proliferation of non-focus-forming cells relative to focus-forming cells. (iii) The reproductive selection described in (ii) cannot completely account for the focus-enhancing effects of low serum. Evidence for point (iii) is drawn from focus-forming assays, in 2% CS, for a clone of cells maintained in 10% CS before assay. Cells from this clone made no foci in 50 assays, performed on 5 x 10(6) cells, over a 21 week period. However, maintenance of those same cells in 2% CS for as little as 2 days before assay caused these consistent non-focus formers to make foci. We conclude that the basis for this progression to the focus-forming phenotype is an adaptation to the metabolically constraining 2% CS environment. We propose that this adaptation results from progressive selection of physiological states.


Expression of alpha, mu and pi class glutathione S-transferases in oval and ductal cells in liver of rats placed on a choline-deficient, ethionine-supplemented diet. Expression of the alpha, mu and pi class glutathione S-transferases (GSTs) in hepatocytes, oval cells and ductal cells derived from the livers of rats placed on a choline-deficient, ethionine-supplemented (CDE) diet for 5 weeks was investigated. An overall decrease in the expression of alpha and mu class GSTs and an over-expression of pi class GST was observed in the liver after CDE treatment as indicated by Northern blotting analysis. Massive disruption of the liver with oval cell infiltration in the sinusoids throughout the lobule occurred after 5 weeks CDE treatment. 'Duct-like' structures consisting of oval-like cells (ductal cells) with rounder nuclei and more cytoplasm than oval cells within the sinusoids were also apparent. Immunocytochemical analysis revealed that the altered expression of GST in the whole liver is attributed to a differential expression of alpha, mu and pi class GSTs in the different cell types in the liver, including hepatocytes, oval cells around the portal region and among the sinusoids, and oval-like cells (ductal cells) in the 'duct-like' structures. In vitro studies using purified oval-ductal cells and hepatocyte populations confirmed the differential expression of GSTs in the varying cell populations in situ. The expression of the alpha and mu class GSTs in hepatocytes does not appear to be altered by the CDE diet. Heterogeneity in distribution of pi class GST was observed in the hepatocyte population, some hepatocytes were stained strongly while no staining was observed in others. Oval and ductal cells represent two distinct populations displaying different expression of GSTs. Pi class GST was detected in the majority of oval and ductal cells. Alpha class GST was detected in < 5% of the oval cell population and was found in > 50% of the ductal cell population. In contrast, mu class GST was absent in ductal cells and was present in 24% of oval cells around the portal region. This supports the view that ductal cells are not of bile ductal origin since mu GST is present in normal bile duct epithelial cells. Furthermore the change in expression of GSTs in the liver after CDE treatment is attributed to the large increase in oval and ductal cell populations.


DNA adduct levels in congenic rapid and slow acetylator mouse strains following chronic administration of 4-aminobiphenyl. 4-Aminobiphenyl (4-ABP) is a human and mouse bladder carcinogen. Epidemiological studies have shown that individuals with a slow acetylator phenotype, especially those exposed to high levels of carcinogenic aromatic amines, show an increased susceptibility to bladder cancer. In order to determine if a slow acetylator phenotype results in increased DNA damage, congenic mouse strains C57BL/6J and B6.A-Nat(s), which differ genetically at the acetyltransferase (EC 2.3.1.5) locus as homozygous rapid (Natr/Natr) and homozygous slow (Nat(s)/Nat(s)) acetylators respectively, were continuously administered 4-ABP.HCl (55-300 p.p.m.) in their drinking water for 28 days. The levels of covalently bound N-(deoxyguanosin-8-yl)-4-ABP-DNA adducts, which are believed to be critical for the initiation of tumors, were quantitated in the liver and bladder by 32P-postlabeling analysis. The levels of the hepatic DNA adduct increased with dose in both sexes, but were independent of the mouse acetylator genotype. At comparable doses, however, the levels of DNA adducts were 2-fold higher in the liver of the female as compared to the male animals. The DNA adducts also increased with dose in bladder of the male mice, but in contrast to the liver, the adduct levels were approximately 2-fold lower in the bladder DNA of the female mice. Also in contrast to the liver, the levels of bladder DNA adducts were significantly higher (P < or = 0.03) in the phenotypic rapid acetylator females compared to the slow acetylators at both 75 and 150 p.p.m. doses; the median levels of adducts were 10-20% higher in the phenotypic slow acetylator male bladders compared to their rapid acetylator counterparts. The results of these studies are consistent with the increased carcinogenicity of 4-ABP to the liver of female mice and the bladder of male mice. They further suggest that factors other than acetylator phenotype limit the extent of DNA adduct formation from 4-ABP in these mice.


Absence of mutations in the functional parts of the p120-GAP gene in carcinogen-induced mouse liver tumors. The GTPase activating proteins (GAP) stimulate the intrinsic GTPase activity of ras-p21 thus converting the protein into its inactive form. We have now analyzed carcinogen-induced mouse liver tumors for the possible occurrence of mutational changes in one of the two known GAP genes, namely p120-GAP. RNA from a total of 21 tumors was reverse transcribed by use of GAP-specific primers, amplified by PCR and sequenced. All known functional domains of p120-GAP were included into the analysis. None of the liver tumors analyzed was found to be mutated within these regions of the gene. Moreover, Southern blot analysis of the gene did not reveal any structural changes. However, at five positions we discovered deviations from the published mouse fibroblast sequence, including two mouse strain-specific sequence polymorphisms.


Tumour promoting phorbol esters activate phospholipase D in mouse skin. Phospholipase D catalyses a transphosphatidylation reaction in the presence of primary alcohols, resulting in the formation of phosphatidylalcohol derivatives. In the present work we show that application of phorbol esters and butanol to mouse skin causes the rapid accumulation of phosphatidylbutanol (PBol), indicating the activation of phospholipase D. A similar accumulation of PBol was observed when the skin was treated with phorbol esters in vivo and skin pieces incubated with butanol in vitro. PBol formation was stimulated by the active tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein and phorbol-12,13-didecanoate (PDD) but not by the inactive promoter 4 alpha-PDD. Accumulation of PBol was not observed 24 h after application of TPA, a treatment which has been shown to deplete epidermal protein kinase C activity.


Formation of 7-(4-oxobutyl)guanine in hepatic DNA of rats treated with N-nitrosopyrrolidine. We have reported previously the formation of two structurally distinct exocyclic guanine adducts (adducts 1 and 6) in liver DNA of F344 rats treated with N-nitrosopyrrolidine (NPYR). In this study, we detected and characterized a previously unidentified guanine adduct in liver DNA of NPYR-treated rats. The structure of this adduct was established as 7-(4-oxobutyl)guanine (adduct 2) by comparison with the synthetic standard and confirmed by NaBH4 reduction to 7-(4-hydroxybutyl)guanine. The level of adduct 2 in liver DNA of F344 rats treated with 450 mg/kg of NPYR by i.p. administration was 643 +/- 9 mumol/mol guanine, approximately one-third of the level of adduct 1. This study is the first to demonstrate the in vivo formation of a formylalkyl-substituted guanine adduct by a nitrosamine.


Cell-transforming potential of low-density lipoproteins. Arterial smooth muscle cell proliferation is thought to be essential for the development of atherosclerotic lesions. The monoclonal hypothesis of atherogenesis proposes that the proliferative smooth muscle cells are derived from a stable transformed cell population. The present study demonstrates for the first time evidence that, in addition to carcinogens such as 3-methylcholanthrene (MCA), 'atherogenic' low-density lipoproteins (LDL) also possess cell-transforming potential. LDL caused dose-dependent cytotoxicity and transformation of C3H/10T1/2 cells of type II and type III morphology of up to six transformed cell clones per 10(4) survivors in the concentration range of 50-200 micrograms cholesterol/ml after 72 h treatments. MCA (0.1 micrograms/ml) induced morphological transformation of 2.6 foci per 10(4) survivors. In a two-stage in vitro transformation assay LDL (5-25 micrograms cholesterol/ml) enhanced MCA-induced cell transformation 2- to 2.4-fold in a dose-dependent way. 'Non-atherogenic' high-density lipoproteins did not induce cell transformation by themselves or in an initiation-promotion model. These results show that LDL could act as (co)carcinogens.


Search for Ha-ras codon 61 mutations in liver tumours caused by hexachlorobenzene and Aroclor 1254 in C57BL/10ScSn mice with iron overload. C57BL/10ScSn mice administered iron--dextran and fed the environmental pollutants hexachlorobenzene (HCB) and polychlorinated biphenyls (PCBs) develop hepatic nodules and carcinomas within 18 months. A range of lesions from the livers were analysed for the presence of mutations in the Ha-ras proto-oncogene at codon 61 using the polymerase chain reaction to amplify DNA from formalin-fixed sections, followed by oligonucleotide hybridization. Only two mutations from 23 preneoplastic and neoplastic lesions induced by HCB were detected (a focus of altered cells and a trabecular cell carcinoma). With Aroclor 1254 no mutations were detected in 28 areas at various stages of carcinogenesis analysed. Sequencing of the two mutations generated by HCB showed a C-->T transversion at the first base of codon 61 (carcinoma) and an A-->T transversion at the second base (proliferative focus). Thus, in marked contrast to some other systems of mouse liver tumour induction, hepatocarcinogenesis caused by HCB and PCBs in C57BL/10ScSn mice is an example of carcinogenesis which does not involve a high frequency of Ha-ras gene mutation at codon 61.


Inhibition of benzo[a]pyrene-induced tumorigenesis by myristicin, a volatile aroma constituent of parsley leaf oil. Glutathione S-transferase (GST) assay-guided fractionation of parsley leaf oil from the edible plant Petroselinum sativum Hoffm. (Umbelliferae) led to the isolation of myristicin. Myristicin showed high activity as an inducer of the detoxifying enzyme GST in the liver and small intestinal mucosa of female A/J mice. Reduction of myristicin yielded dihydromyristicin that retained the GST-inducing activity. Myristicin and dihydromyristicin were tested for their capacity to inhibit benzo[a]pyrene (B[a]P)-induced tumor formation in female A/J mice. A 65% inhibition of the tumor multiplicity in the lung was observed as the result of treatment of myristicin. Dihydromyristicin produced small or insignificant reduction of lung tumor formation. In the forestomach, myristicin showed a 31% inhibition of tumor formation; while dihydromyristicin exhibited a 27% inhibition. Comparison of the structures and activities indicated that the saturation of the isolated double bond in myristicin resulted in a significant decrease in the inhibitory activity against B[a]P-induced tumorigenesis. The present results showed that myristicin, an active inducer of GST activity, is an effective inhibitor of B[a]P-induced tumorigenesis in mice. Stimulation of GST activity by myristicin could be a major mechanism for its inhibition of B[a]P or other carcinogens that may be detoxified in the same manner. As a culinary herb parsley is regularly consumed by humans. Parsley leaf oil is also used extensively for garnishing and seasoning. The results of this study indicate that as a major volatile aroma constituent of parsley myristicin may be an effective cancer chemopreventive agent.


Reversal of food restriction-induced inhibition of mouse skin tumor promotion by adrenalectomy. Restricting the food intake of laboratory mice and rats markedly reduces the incidence of spontaneous and experimentally induced cancers. Using the two-stage skin tumorigenesis model in CD-1 mice, we report now that food restriction suppresses 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of epidermal [3H]thymidine incorporation as well as TPA promotion of skin papillomas, whereas adrenalectomy completely reverses the inhibition in [3H]thymidine incorporation and tumor development. These results suggest that the adrenal gland may play an important role in mediating the tumor inhibitory effect of food restriction.


Mechanisms of the enhanced liver carcinogenesis by choline in female rats: delay in liver growth after partial hepatectomy and stimulation of 2-AAF mitoinhibition. Choline given to female rats resulted in an enhanced growth of focal lesions induced by the Solt-Farber model [diethylnitrosamine (DEN) as initiator and 2-acetylaminofluorene (2-AAF) associated with partial hepatectomy (PH) as promoter] when it was administered during promotion, but not during DEN treatment. Based on these data, the mechanisms by which choline might interfere with 2-AAF-PH promotion have been investigated. Compensatory liver growth after PH was faster in females than in males as shown by the restoration of both liver weight and protein content in rats fed a basal diet or a 2-AAF diet. The [3H]thymidine incorporation into DNA occurred a few hours earlier in females than in males. Choline administered to normal females delayed the peak of liver regeneration, by inhibiting [3H]thymidine incorporation into DNA and the mitotic index, resulting in later restoration of liver mass. 2-AAF feeding for 1 week markedly depressed the incorporation of precursor into DNA following PH in both sexes but at greater extent in males. Choline enhanced the mitoinhibition of 2-AAF only in females. No effect of choline was observed in male rats either with or without 2-AAF. The differential effect of choline between sexes in the development of focal lesions appeared to be related to the sexual dimorphism in the 2-AAF mitoinhibition and in the liver proliferation after PH.


Mammary carcinoma suppressor and susceptibility genes in the Wistar-Kyoto rat. The Copenhagen (Cop) rat carries a homozygous mammary carcinoma suppressor (MCS) gene that prevents both spontaneous and induced mammary cancer. Here we identify an additional rat strain, the Wistar-Kyoto (WKy), that is resistant to the development of mammary carcinomas. This rat strain is similar to Cop in having one homozygous copy of a mammary suppressor gene. The Cop MCS gene and the gene responsible for mammary cancer resistance in the WKy were found to be very closely linked genetically and thus are likely to be the same gene. It was found that, unlike the Cop strain, the WKy strain also carries multiple copies of a susceptibility gene(s). This is analogous to the susceptible Wistar-Furth strain, which carries three copies of dominant susceptibility genes. The MCS gene is epistatic in regard to these susceptibility genes. The availability of the WKy rat strain carrying MCS on a background independent of the Cop strain will aid both mechanistic studies and the molecular cloning of MCS.


Peroxidative activation of o-phenylhydroquinone leads to the formation of DNA adducts in HL-60 cells. Using 32P-postlabeling we studied DNA adduct formation in HL-60 cells treated with the o-phenylphenol metabolites o-phenylhydroquinone (o-PHQ) and o-phenylbenzoquinone (o-PBQ). Treatment with 25-500 microM o-PHQ for 8 h produced one principal and three minor adducts with a relative distribution of 80, 10, 6 and 4%. The relative adduct levels from these treatments were 0.26-2.31 adducts/10(7) nucleotides. Treatment with 25-250 microM o-PBQ for 2 h resulted in a similar level of DNA modification and adduct distribution. Reaction of purified calf thymus DNA with o-PBQ produced one DNA adduct, which did not correspond to the major adduct produced in HL-60 cells. These results show that o-PHQ and o-PBQ can form DNA adducts. Peroxidase activation of o-phenylphenol may therefore play a role in the carcinogenic effect of this compound.


High expression of manganese superoxide dismutase in 7,12-dimethylbenz[a]anthracene-induced ovarian cancer and increased serum levels in the tumor-bearing rats. Manganese superoxide dismutase (Mn-SOD) is strongly expressed in human ovarian cancer, and the serum level of the enzyme is a useful marker for the diagnosis and monitoring of human ovarian cancer. In the present study we found that Mn-SOD was highly expressed in primary and transplanted ovarian cancers in rats induced by 7,12-dimethylbenz[a]anthracene (DMBA), as judged by enzyme-linked immunosorbent assay as well as by Northern blot analysis. The serum levels of Mn-SOD in the tumor-bearing rats were also higher than those in control rats. The antibody strongly reacted with rat ovarian carcinoma tissues. These data suggest that DMBA-induced ovarian cancer in rats is a good experimental model for human ovarian cancer, and that Mn-SOD is also a good marker for disease in the animal model.


7-Methylguanine levels in DNA of smokers' and non-smokers' total white blood cells, granulocytes and lymphocytes. The effect of smoking was investigated on the formation of 7-methylguanines in human peripheral white blood cells. DNA was isolated from total white blood cells, granulocytes and lymphocytes from 10 smokers and 10 non-smokers. 32P-Postlabeling was performed by using anion-exchange chromatography enrichment of adducts. In smokers the mean DNA adduct levels were 6.9, 4.7 and 23.6 7-methylguanine residues/10(7) nucleotides in total white blood cells, granulocytes and lymphocytes respectively. The corresponding values in non-smokers were 3.4, 2.8 and 13.5 adducts/10(7) nucleotides. The mean adduct level was significantly higher in lymphocytes than in total white blood cells or granulocytes both in smokers and in non-smokers. The mean adduct levels differed significantly between smokers and non-smokers.


A study of tobacco carcinogenesis XLVIII. Carcinogenicity of N'-nitrosonornicotine in mink (Mustela vison). During tobacco processing and smoking, nicotine and nornicotine give rise to N'-nitrosonornicotine (NNN), a highly abundant, strong carcinogen. NNN is known to exert carcinogenic activity in mice, rats and hamsters. Major target organs for NNN carcinogenicity in the rat are the esophagus and the nasal mucosa, and in the Syrian golden hamster trachea and nasal mucosa. In comparison with the rat, the mink (Mustela vison) has a markedly expanded nasal mucosa. Therefore, we explored in this study whether the mink could serve as a non-rodent model for nasal carcinogenesis using NNN as the carcinogen. Twenty random-bred mink, beginning at the age of 3 weeks, received twice weekly s.c. injections of NNN, a total dose of 11.9 mM per animal over a 38 week period. All of the 19 mink at risk developed malignant tumors of both the respiratory and the olfactory region of the nose within 3.5 years. In most animals the malignant tumors, primarily esthesioneuroepithelioma, invaded the brain. Remarkably, NNN induced no other tumors in the mink. None of the control animals developed nasal tumors nor tumors at other sites during the 3.5 years of the assay. The historical data from the farm did not reveal any spontaneous occurrence of nasal tumors in mink at any age. This study supports the concept that NNN is a proven carcinogen for multiple species of mammals and that the mink can serve as a non-rodent, non-inbred animal model for nasal carcinogenesis, especially since NNN induces only tumors in the nasal cavity in this species and not at other sites, as it does in mice, rats and hamsters.


Biliary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the rat. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pancreas carcinogen in rats. The biliary excretion of NNK was therefore studied in anesthetized female Sprague-Dawley rats following i.p. administration of 0.7 mumol/kg [carbonyl-14C]NNK. The concentration of radioactivity peaked within 30 min and decreased thereafter exponentially. Cumulative excretion of radioactivity reached a plateau at 6-9% of the total dose. HPLC analysis revealed the presence of 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid), 4-oxo-4-(3-pyridyl)-butyric acid (keto acid), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butyl beta-D-glucopyranosiduronic acid (NNAL Glu), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and NNK. NNAL Glu was the major metabolite contributing 34 +/- 4% of total radioactivity in bile at 30 min and 58 +/- 4% at 5 h. The percentage of acidic metabolites remained constant at approximately 20%. In contrast, the percentage of NNK and NNAL decreased within the first 2 h to < 5% and < 10% respectively. The elimination kinetics of NNK and its metabolites fitted into a one-compartment model with a half-life of 37 min for NNK, 52 min for NNAL and 110 min for NNAL Glu and acidic metabolites. In three rats dosed with 240 mumol/kg NNK i.p., the concentration of radioactivity peaked after 1-2 h and decreased very slowly thereafter. After 5-8 h a total of 12-17% of the dose has been excreted in the bile with no indication of a plateau. At all time points NNAL Glu was the major metabolite contributing up to 95% of total radioactivity in bile. The percentage of acidic metabolites was < 5% throughout the experiment. Whereas NNK contributed one-third of the radioactivity at 30 min and decreased rapidly, the percentage of NNAL in bile remained rather constant at approximately 5-10%. In conclusion, the detection of NNK, NNAL and NNAL Glu gives support to the hypothesis that tobacco-specific carcinogens could reach the pancreas retrograde from the bile, especially at high NNK concentrations.


Repair of mitochondrial DNA after various types of DNA damage in Chinese hamster ovary cells. Using methodology recently developed to assess gene-specific DNA repair, we have demonstrated that it is possible not only to study mitochondrial DNA repair, but also directly to compare mitochondrial and nuclear DNA repair in the same biological sample. Complex enzymatic mechanisms recognize and repair nuclear DNA damage, but it has long been thought that there was no DNA repair in mitochondria. Therefore, in an attempt to delineate more clearly which DNA repair mechanisms, if any, are functioning in mitochondria, we have investigated the repair of several specific DNA lesions in mitochondrial DNA. They include cyclobutane dimers, cisplatin intrastrand adducts, cisplatin interstrand crosslinks and alkali-labile sites. We find that pyrimidine dimers and complex alkylation damage are not repaired in mitochondrial DNA, and that there is minimal repair of cisplatin intrastrand crosslinks. In contrast, there is efficient repair of cisplatin interstrand crosslinks as evidenced by approximately 70% of the lesions being removed by 24 h. Additionally, there is efficient repair of N-methylpurines following exposure to methylnitrosourea with approximately 70% of the lesions being removed by 24 h. The results of these studies reveal that repair capacity of mitochondrial DNA damage depends upon the type of lesion produced by the damaging agent. We speculate that a process similar to the base excision mechanism for nuclear DNA exists for mitochondrial DNA but that there is no nucleotide excision repair mechanism to remove more bulky lesions in this organelle.


Preferential and strand-specific DNA repair of (6-4) photoproducts detected by a photochemical method in the hamster DHFR gene. A non-enzymatic method that was previously shown to create single-strand DNA breaks at the location of (6-4) photoproducts in the overall genome was adapted to measure (6-4) photoproducts at the level of the gene. This approach employs a photoisomerization step that converts the (6-4) photoproduct into the Dewar valence photoisomer, which is more alkaline labile and allows for the creation of a single-strand DNA break at this site. These strand breaks were quantified via Southern analysis and the DNA repair of (6-4) photoproducts was measured over 2, 4 and 8 h after a UV dose of 40 J/m2. A comparison of repair efficiency in the actively transcribed dihydrofolate reductase (DHFR) gene, a transcriptionally inactive genomic region and the overall genome (as measured by radioimmunoassay) showed preferential repair of the active gene. The active DHFR gene showed 59% repair by 8 h compared to 33% repair in the inactive downstream region. Analysis of (6-4) photoproduct repair in the transcribed and non-transcribed strands of the DHFR gene indicate some strand specificity with 62% repair in the transcribed strand at 8 h compared with 43% repair in the non-transcribed strand. However, this strand bias is much less distinct than has been reported for the major UV photoproduct, the cyclobutane pyrimidine dimer.


Overexpression of human DNA repair protein N-methylpurine-DNA glycosylase results in the increased removal of N-methylpurines in DNA without a concomitant increase in resistance to alkylating agents in Chinese hamster ovary cells. N-Alkylpurines induced in DNA by simple monofunctional alkylating agents are known to be cytotoxic and possibly indirectly mutagenic. These adducts are removed by the ubiquitous N-methylpurine-DNA glycosylase (MPG) in a multistep repair pathway. Chinese hamster ovary (CHO) cell clones expressing 2- to 16-fold enhanced levels of MPG activity were isolated from cells stably transfected with human MPG cDNA expression plasmids. The in vivo removal of 3-methyladenine and 7-methylguanine from some of these lines was analyzed and was observed to reflect their MPG levels. These cell lines did not develop increased resistance, as compared to the control, in regards to cytotoxic, mutagenic and sister chromatid exchange inducing effects of the alkylating agents that induce 3-alkyladenine and 7-alkylguanine as the major alkyl adducts in DNA. These results suggest that the MPG activity is not limiting in the multi-step repair pathway of N-alkylpurines in CHO cells.


Selective redistribution of protein kinase C isozymes by thapsigargin and staurosporine. Protein kinase C (PKC) is the major cellular receptor for tumor promoting phorbol esters. Phorbol esters activate alpha-, beta-, delta- and epsilon-PKCs in GH4C1 rat pituitary cells and cause their redistribution from a soluble to a particulate fraction. We have now characterized the effect of several non-phorbol ester tumor promoters on PKC isozyme distribution in GH4C1 cells. The incomplete tumor promoter mezerein caused redistribution of alpha-, beta-, delta- and epsilon-PKCs. Thus, it did not display partial agonist activity. The phosphatase inhibitor okadaic acid did not cause redistribution of any isozyme. The calcium ATPase inhibitor thapsigargin and the ser/thr kinase inhibitor staurosporine caused redistribution of epsilon-PKC and, to a lesser extent, delta-PKC. Although the mechanism of the selective effect on delta- and epsilon-PKCs is not yet known, these data clearly demonstrate that their subcellular distribution can be regulated by a pathway that does not influence alpha- and beta-PKCs. Phorbol ester activation of epsilon-PKC was associated with appearance of a more slowly migrating immunoreactive band in the particulate fraction. Both epsilon-PKC forms accumulated phosphate during phorbol ester treatment. The phosphorylated forms of epsilon-PKC were preferentially recovered in the particulate fraction. Although staurosporine caused redistribution, it prevented the phorbol dibutyrate (PDBu)-mediated appearance of the upper band of the doublet and the increased phosphorylation of both bands. The PDBu-mediated redistribution of alpha- and beta-PKCs was not inhibited by staurosporine, even though staurosporine effectively inhibited PKC catalytic activity. Therefore, catalytic activity is not required for redistribution.


Formation of 8-hydroxydeoxyguanosine within DNA of mouse keratinocytes exposed in culture to UVB and H2O2. Exposure to UV light contributes to the development of skin cancer. The importance of reactive oxygen species in UV-radiation carcinogenesis has been recognized for some time and several associated DNA base modifications have been identified. In particular, 8-hydroxydeoxyguanosine (8-OHdG) has been well studied as an indicator of oxidative damage to calf thymus DNA exposed to a variety of oxygen-generating systems, including UV light. However, to date, few studies of 8-OHdG have been conducted in cell or animal systems and those in vitro investigations that studied UV exposure have used UVC (< 290 nm), not the UVB (290-320 nm) or UVA (320-400 nm) ranges to which organisms are exposed through sunlight. The objective of this study was to measure 8-OHdG formation in the DNA of cultured mouse keratinocytes exposed to UVB. Using HPLC with electrochemical detection, background levels of 8-OHdG were approximately 6 fmol/micrograms DNA in DNA isolated and digested to the nucleoside level. UVB induced 8-OHdG up to 100% above that for mock-treated cells at a dose of 630 mJ/cm2 (dose-response range: 210-630 mJ/cm2). UVB exposure at 630 mJ/cm2 combined with 5 mM H2O2 elevated 8-OHdG formation up to 280% above that in control cells, whereas H2O2 alone had no effect. These results suggest that factors which increase the generation of reactive oxygen species by UV light may be potent cofactors of UV-radiation carcinogenesis.


32P-postlabeling analysis of the formation and persistence of DNA adducts in mammary glands of parous and nulliparous mice treated with benzo[a]pyrene. The susceptibility of the rodent mammary gland to known chemical carcinogens can vary with the stage of gland development. Full-term pregnancy (parity) can confer permanent structural and functional changes in the gland that are associated with decreased breast cancer risk in humans and protection from mammary carcinogenesis in rodents. In this study, the formation and persistence of benzo[a]pyrene (B[a]P)-derived DNA adducts in vivo was determined in the abdominal mammary organs of adult nulliparous and parous BALB/c mice treated orally with the carcinogen. Mammary DNA isolated from animals in both groups revealed only one major adduct on TLC maps by P1-nuclease 32P-postlabeling analysis. The major adduct co-migrated with the (+)-enantiomer of anti-BPDE-dGp. Much lower levels of the (-)-enantiomer were detected. Most of the adduct quantitated was probably contributed by cells of the stromal compartment, since the gland-free organ (cleared fat pad) generated essentially equivalent profiles and level of adduct by 32P-postlabeling. Comparable levels of the B[a]P-derived adduct were observed in the intact mammary organ of both nulliparous and parous mice during a time course from 1 to 28 days after treatment. In both cases, adduct removal occurred exponentially with a half-life of approximately 16 days. The capacity for de novo formation of reactive metabolites by the mammary organ was demonstrated in vitro: digests of DNA from mammary mince exposed to B[a]P generated an adduct on TLC maps that also co-migrated with the (+)-anti-BPDE-dGp standard. Thus, our cumulative findings confirm the capacity of mammary cells to form potentially carcinogenic DNA adducts; however, the functional changes that occur in the mouse mammary gland as a result of parity did not influence the profile, level or persistence of adduct following exposure to a known carcinogen. Other factors, such as changes in mammary cell cycle kinetics or responsiveness to promotional stimuli may be more causally related to reduction in incidence of neoplasia observed in parous animals.


Purification and characterization of rat hepatic microsomal N,O-acyltransferases. Hepatic microsomes of female F344 rats were capable of N,O-acyltransfer of N-hydroxy-N-acetyl-2-aminofluorene (N-OH-AAF) and N-hydroxy-N-formyl-2-aminofluorene (N-OH-FAF), N-deacetylation of N-OH-AAF and N-acetyl-2-aminofluorene (2-AAF), and O-deacetylation of 4-nitrophenyl acetate (NPA). The activity for N,O-acyltransfer of N-OH-FAF was approximately 20 times greater than that of N-OH-AAF. These microsomal activities were inducible by phenobarbital and were inhibitible by paraoxon. Four distinct N,O-acyltransferases were purified from solubilized hepatic microsomes of phenobarbital pretreated rats. These enzymes were purified to homogeneity, as judged by SDS-PAGE and analytical IEF. Their pIs were approximately 5.0, 5.5, 6.0 and 6.5 and their mol. wts were approximately 60, 61, 180 (a homotrimer of 59 kDa) and 60 kDa respectively. All the enzymes catalyzed the N,O-acyltransfer of N-OH-FAF, the N-deacetylation of N-OH-AAF and 2-AAF and the O-deacetylation of NPA. Among these four enzymes, the hydrolysis of NPA was best catalyzed by pI 6.5 protein, of 2-AAF by the pI 5.5 protein, and of N-OH-AAF by the pI 5.0 protein. The pI 5.5 and pI 6.5 proteins were equally active for N,O-acyltransferase and were more active than the other enzymes. The present study demonstrates that rat hepatic microsomal activities of N,O-acyltransfer, N-deacetylation and O-deacetylation are attributable to the same enzymes.


Transformation of non-tumorigenic osteoblast-like human osteosarcoma cells by hexavalent chromates: alteration of morphology, induction of anchorage-independence and proteolytic function. Soluble chromium (VI) compounds either alone or in combination with 3-methylcholanthrene (MC) were used to transform non-tumorigenic osteoblast-like human osteosarcoma cells (HOS TE85). The Cr(VI) compounds were highly toxic to these cells with LC50 values in the range of approximately 0.5-1.0 microM. Continuous passaging of the treated cells resulted in sustained increase in anchorage-independent (AI) colony formation. Treatment with Cr(VI) and MC resulted in substantial increase in AI growth. At the XVth passage, a number of individual AI colonies were expanded in culture and used for further studies. The cells are refractory in appearance and grow as 'nests' rather than as monolayers. The cell lines have relatively high plating efficiency (PE) in soft agar and respond to promotional effect of phorbol-12-myristate-13-acetate by an increase in PE and in the size and number of AI colonies. While the isolated cells are not tumorigenic when tested in athymic nude mice, most of the lines possess higher levels of plasminogen activator (PA) activity, considered as one of the markers of transformation. This is also reflected in the increase in the steady state level of urokinase type PA mRNA. These results show that Cr(VI) compounds are capable of promoting human cells to an altered phenotype characteristic of a stage in the carcinogenesis cascade.


Aflatoxin toxicity in cultured human epidermal cells: stimulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Aflatoxin B1 (AFB1) at 1 microgram/ml was markedly toxic to human epidermal cells grown in the Rheinwald-Green 3T3 feeder layer system. At 0.1 microgram/ml, the toxicity was barely evident, as assessed by colony expansion during a 2 week exposure, but it was dramatically stimulated by 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which was non-toxic alone. Neither AFB1-dihydrodiol nor AFB2 were toxic in the presence or absence of TCDD, indicating that metabolism to the 8,9-epoxide was responsible for the AFB1 toxicity. Stimulation of AFB1 epoxidation by TCDD was also indicated by the > 20-fold increase in DNA adduct formation in cultures exposed to [14C]AFB1 and TCDD for 4 days as compared to AFB1 alone. Analysis of free metabolites in culture medium by reverse-phase HPLC revealed that confluent epidermal cultures metabolized AFB1 to AFM1, AFB2a and aflatoxicol. In the presence of TCDD, the levels of AFM1 were higher (14 versus 3% of dose) as were those of AFB2a (3 versus 0.5% of dose), while aflatoxicol levels were lower (0.8 versus 2% of dose). In the absence of irradiated 3T3, the toxicities of AFB1, AFB2, AFM1 and aflatoxicol to cells in serum-free medium (0.15 mM Ca2+) were similar to those in the feeder layer system. Although this moderately low calcium concentration appeared quite satisfactory for observing toxicity, the response was attenuated at a lower calcium concentration (0.09 mM Ca2+).


Cigarette smoking related polycyclic aromatic hydrocarbon-DNA adducts in peripheral mononuclear cells. Studies on cigarette smoking related polycyclic aromatic hydrocarbon-DNA adducts in blood have produced conflicting results. To determine whether a subset of specific white blood cells is a useful marker for monitoring exposure to cigarette smoke, blood was obtained from 63 heavy smokers and 27 non-smokers. Adduct levels were determined by competitive enzyme-linked immunosorbent assay with a polyclonal antiserum recognizing benzo[a]pyrene and structurally related diolepoxide-DNA adducts. Analysis of the lymphocyte plus monocyte fraction from smokers indicated 70% had detectable adducts with a mean of 4.38 +/- 4.29 adducts/10(8) nucleotides, while in non-smokers the corresponding values were 22% and 1.35 +/- 0.78/10(8) (P < 0.001). Plasma cotinine levels differed significantly in smokers (286 +/- 90 micrograms/l) compared to non-smokers (4.4 +/- 3.3 micrograms/l) (P < 0.001). However, cotinine was not correlated with self-reported smoking history in these heavy smokers. Nor were DNA adducts in smokers correlated with cigarettes per day, pack-years and plasma cotinine, indicating large interindividual variation in DNA adduct formation. These data demonstrate lymphocytes plus monocytes from smokers have elevated levels of polycyclic aromatic hydrocarbon diolepoxide-DNA adduct levels compared to non-smokers.


Promoting effects of both dietary cholesterol and cholestyramine on pancreatic carcinogenesis initiated by N-nitrosobis(2-oxopropyl)amine in Syrian golden hamsters. The effects of dietary cholesterol and cholestyramine on pancreatic carcinogenesis initiated with N-nitrosobis(2-oxopropyl)amine (BOP) were investigated in 120 female Syrian golden hamsters. BOP (70 mg/kg body weight) was injected s.c. once at the beginning of the experiment. Starting 2 weeks later, the animals were then maintained on basal diet or diets containing either 0.5% cholesterol or 1% cholestyramine for a further 16 weeks. All surviving hamsters were killed at week 18, and the pancreas tissues examined histologically. The incidences of pancreatic carcinomas in hamsters fed cholesterol and the cholestyramine supplement were 40.0 and 30.0% respectively; in both cases significantly higher than the 6.9% incidence in the basal diet group. Cholesterol contents of the serum, pancreas and liver were significantly increased by cholesterol feeding and significantly decreased by the cholestyramine diet. The cholesterol diet also significantly increased pancreatic protein and DNA contents, and the concentration of total bile acids and the level of lithocholic acid in gallbladder bile. The cholestyramine diet significantly increased total pancreatic DNA and protein contents, and pancreatic weight. The results thus indicated that both dietary cholesterol and cholestyramine can enhance BOP-initiated pancreatic carcinogenesis in hamsters.


Mutational specificity of chromium(VI) compounds in the hprt locus of Chinese hamster ovary-K1 cells. Chromium(VI) compounds exert their genotoxicity and mutagenicity by complex metabolic reducing pathways that generate a variety of reactive forms of chromium and free radicals. To investigate the molecular nature of chromium-induced mutations, we characterized the entire coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene of 27 independent mutants derived from chromium(VI) oxide (CrO3)-treated Chinese hamster ovary-K1 cells, by direct sequencing of PCR-amplified cDNA. Among these mutants, 10 consisted of single base substitutions, five contained two base substitutions, one had four base substitutions, six were splicing mutations, and five exhibited single base pair insertions or deletions. All of the base substitutions and most of the frameshift mutations observed were located at A/T-rich sequences. More than 90% of the base substitutions (22/24) occurred in A.T base pairs. Among them, T-->A and T-->G transversions (18/22) predominated. The mutational hotspots for single and double base substitutions were the 3' thymidine of 5'PuT and thymidines of 5'ATTT sequences respectively. This mutational specificity was also observed in CHO-K1 cells treated with two other chromium(VI) compounds, namely K2Cr2O7 and PbCrO4. Strand bias was noticed in chromium mutagenicity, since 77% of T base substitutions occurred on the non-transcribed strand. This highly sequence-specific mutation spectrum suggests that a particular form of chromium may directly interact with DNA at these hotspot sequences.


Response of the ke test to NCI/NTP-screened chemicals. III. Complementary value of ke in screening for carcinogens. The value of using a physico-chemical carcinogen-screening test, the ke test, in conjunction with the Salmonella typhimurium/microsome assay (the Ames test) and/or structural alerts of reactivity (the S/A test), is analyzed on the basis of the response of the three tests to 171 chemicals of known rodent carcinogenicity. The Ames test is widely used to screen chemicals for potential carcinogenicity; however, its relatively low sensitivity (proportion of true positives among carcinogens tested) has prompted a search for complementary tests that increase sensitivity without an unacceptable decrease in specificity (proportion of true negatives among non-carcinogens tested). The S/A test is a structural analysis based on recognition of chemicals groups likely to react with DNA. The S/A test does not complement the Ames test well, because of the high similarity of responses (dependence) between these two tests. The ke test measures the affinity of a test chemical for electrons, and has a sensitivity and specificity comparable to the Ames test. The ke test is shown in this work to complement both the Ames test and the S/A test. Addition of the ke test to either the Ames test or the S/A test results in a substantial decrease in false negatives and an approximately equal increase in false positives, which is a trade-off that would be desirable in all but the least risk averse situations. The S/A and ke battery has a sensitivity of > 0.9, and could be applied to untested chemicals without any biological testing. In view of these observations, it is proposed that the ke test be considered in developing future strategies to optimize the screening of potential carcinogens in the most cost-effective manner.


Molecular dynamics of alkylated DNA.

Molecular dynamics of alkylated DNA. The effect of methylation of the O4 atom of thymine in two oligonucleotide sequences is investigated by molecular dynamics simulations. Three types of environments are considered including: (i) in vacuo calculation, with a distance-dependent dielectric function and unhydrated counter-ions; (ii) in vacuo calculation, with a distance-dependent dielectric constant and hydrated counter-ions; and (iii) with a 9 A thick explicit water layer and counter-ions. In all environments, the oligonucleotide sequence containing the chemically modified thymine paired with guanine is more stable than the oligonucleotide sequence in which the modified thymine is paired with adenine. The methyl group attached to the O4 atom of thymine is found in a syn configuration with respect to the N3 atom. The best fit between the experimental NMR results and the molecular dynamics simulations is obtained using the environment with hydrated counter-ions.


Inhibition of mouse skin tumorigenesis by dexamethasone occurs through a Ha-ras-independent mechanism. The Ha-ras oncogene has been shown to be point-mutated and overexpressed in papillomas induced by the two-stage skin tumorigenesis regimen of 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Glucocorticoids inhibit mouse skin tumorigenesis when applied with the initiation agent or with the promoting agent. We have extended these studies to evaluate whether dexamethasone (Dex) treatment could inhibit development of already established tumors. An additional objective of this study was to investigate whether glucocorticoids directly inhibit Ha-ras gene expression at the level of transcription during skin tumorigenesis. In this report we demonstrate that topical Dex treatments significantly suppressed the formation of additional tumors relative to the acetone control group. However, Northern blot analysis of total RNA isolated from representative tumors during a series of sequential weeks of promotion indicated that Dex did not have a direct effect on Ha-ras steady-state mRNA levels despite the decrease in additional tumor numbers in the Dex-treated groups. We also investigated short-term effects of Dex on endogenous Ha-ras expression in normal mouse epidermis. Topical Dex administration had no effect on endogenous Ha-ras steady-state mRNA levels in normal skin after 2 or 24 h. To ensure that endogenous corticosterone levels in the SENCAR mouse were not influencing our results, Ha-ras mRNA levels in epidermis from SENCAR mice adrenalectomized 48 h prior to being killed were compared to Ha-ras levels in normal epidermis by Northern blot analysis. The data from this analysis revealed that bilateral adrenalectomy had no effect on Ha-ras steady-state mRNA levels in epidermis compared to ras levels in normal mouse epidermis. In summary, our results demonstrate that although Dex can inhibit further tumor development in DMBA/TPA-treated mouse epidermis, it does not do so by directly effecting Ha-ras gene expression in mouse epidermis.


Evidence for a ras gene mutation in azoxymethane-induced colonic aberrant crypts in Sprague-Dawley rats: earliest recognizable precursor lesions of experimental colon cancer. The main objective of the present investigation was to understand the molecular events involved in the genesis of aberrant crypt foci. Aberrant crypt foci were induced in Sprague-Dawley rats with a single injection of azoxymethane. Aberrant crypts have been identified topographically in the colon and are hypothesized to represent preneoplastic lesions. In order to understand the molecular events involved in the early stages of colon cancer, PCR-amplified DNA from aberrant crypts was hybridized with oligonucleotide probes specific for the detection of point mutations in codon 12 of K-ras. The mutation identified was a G to A transition resulting in the substitution of the amino acid aspartic acid (asp) for glycine (gly). This mutation was present in 6/19 (32%) of aberrant crypts examined. The identical mutation was also identified in adenomacarcinoma tissue while no mutation could be detected in normal intestinal mucosa. For further confirmation of these results, the presence of the mutated ras protein (rasAsp-12) was detected in aberrant crypts by immunohistochemistry. This investigation provides the first identification of a ras point mutation in aberrant crypt foci.


Mechanism of formation of the thioether conjugate of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT). The formation of thioether conjugates is an important pathway for inactivation of certain carcinogens. This study assessed the mechanism by which the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT) forms a glutathione conjugate (ANFT-SG). Peroxidatic metabolism of ANFT, in the presence of glutathione, results in ANFT-SG formation. Both prostaglandin H synthase and horseradish peroxidase can catalyze this reaction. Metabolism of the reducing co-substrates ANFT, phenol, and aminopyrine elicit increases in oxidized glutathione (GSSG). ANFT-SG formation is potentiated by phenol and aminopyrine. tert-Nitrosobutane (tNB), a thiyl radical trap, prevented increases in both GSSG and ANFT-SG. Increasing concentrations of ANFT elicited corresponding increases in both GSSG and ANFT-SG. Peroxidatic metabolism of ANFT in the presence of glutathione, but not in the absence of glutathione, resulted in oxygen uptake. The formation of GSSG and oxygen uptake are consistent with the presence of thiyl radicals during ANFT metabolism. 5,5-Dimethyl-1-pyrroline N-oxide, a thiyl radical trap, was not as effective as tNB in inhibiting the formation of ANFT-SG and GSSG. Ascorbic acid, a reducing cosubstrate and antioxidant, was very effective in preventing ANFT-SG and GSSG formation, while the strong nucleophile methionine was ineffective. To clarify effects of different test agents, their effects on aminopyrine cation radical formation were assessed. Results are consistent with ANFT reacting with thiyl radicals to form ANFT-SG. ANFT appears to be a thiyl radical trap. Peroxidatic metabolism of ANFT probably results in the formation of a cation radical rather than a carbon-centered radical.


Reaction kinetics of DNA-histone crosslinking by vinyl acetate and acetaldehyde. The formation and stability of DNA-protein crosslinks (DPXLs) formed by incubation of pUC13 plasmid DNA and calf thymus histones with 1-100 mM acetaldehyde was studied using a filter binding assay. DPXLs were formed at a rate of 127 DPXLs/plasmid molecule/mmol acetaldehyde in a reaction containing 1 microgram of histones and 0.33 microgram of DNA at 37 degrees C for 1 h. Acetaldehyde-induced DPXLs were unstable at 37 degrees C, with loss of up to 75% by 8 h. Crosslink formation was significantly higher at lower pH, with 3- and 2-fold higher levels at pH 5 and 6 respectively than at pH 7.5. Induction of DPXL formation by 1-100 mM vinyl acetate in the presence of rat liver microsomes was observed at 37 degrees C over 3 h. DPXL accumulation followed S-phase enzymatic kinetics, with a rate of formation of 1.1 DPXLs/plasmid molecule/mmol vinyl acetate/microgram microsomal protein/microgram DNA. Vinyl acetate was unable to cause formation of DPXLs in the absence of microsomes. A carboxylesterase inhibitor, bis-(p-nitrophenyl) phosphate, was able to block DPXL formation by vinyl acetate and microsomes. This work supports the hypothesis that DPXL formation by vinyl acetate requires microsomal metabolism to acetaldehyde, which is the active crosslinking agent.


Accumulation of O6- and 7-methylguanine in DNA of N-nitroso-N-methylbenzylamine-treated rats is restricted to non-target organs for N-nitroso-N-methylbenzylamine-induced carcinogenesis. The distribution and accumulation of O6-methylguanine (O6-meGua) and 7-methylguanine (7-meGua) was investigated immunocytochemically in target and non-target tissues of rats injected twice weekly with 0.5 mg N-nitroso-N-methylbenzylamine (NMBzA)/kg for 16.5 weeks. Seventy two hours after every two or three doses, two NMBzA-treated rats and one control rat were killed. Tissue-specific cell proliferation was investigated after two injections of bromodeoxyuridine (BrdU) to rats unexposed to NMBzA. Neither O6-meGua- nor 7-meGua-specific immunostaining could be observed in the target tissues for tumor induction, i.e. esophagus, tongue and forestomach. Accumulation of both O6-meGua and 7-meGua was found, however, in nasal, tracheal and bronchiolar epithelia and glands--tissues for which tumor induction by NMBzA has not been reported. An explanation for this phenomenon might be the relatively low levels of cellular proliferation we observed in the latter epithelia. The present results support the hypothesis that the tumorigenic organotropism of NMBzA is determined both by the level of DNA methylation and the proliferative capacity of the methylated cells.


Characterization of ethanol's enhancement of tumorigenesis by N-nitrosodimethylamine in mice. The concentration-, time- and route-dependent effects of ethanol co-administration on tumorigenesis by N-nitrosodimethylamine (NDMA) were characterized in strain A male mice. With drinking-water administration, 1% ethanol was as effective as 5 or 10% in effecting a 4-fold enhancement of lung tumorigenesis by 5 p.p.m. NDMA. In a study of cumulative effects over time, 10% ethanol given with 1 p.p.m. NDMA resulted in a progressive increase in lung tumors from 16 to 72 weeks. In addition, at 72 weeks, the ethanol co-treatment resulted in a significant increase in kidney adenomas and possibly in vascular tumors of liver. A single i.g. dose of 5 mg/kg NDMA was significantly tumorigenic for lung, and the effect was dose-dependently increased by inclusion of ethanol, for up to a 9-fold enhancement with 20% ethanol. When 10% ethanol was given in the drinking water while NDMA was administered as 20 1 mg/kg doses by other routes--i.g., i.p., s.c. or i.v.--the ethanol treatment was without effect on lung tumor numbers. Collectively, the results provide strong support for inhibition of hepatic first-pass clearance of NDMA by cytochrome P450 2E1 as the mechanism of ethanol's effect, and suggest that several other possible mechanisms are unlikely. They also illustrate that a moderate dose of ethanol cumulatively increases tumor risk from a low dose of NDMA given over most of the lifetime of the animal.


Proliferative dependent regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in human cells. The relationship between the proliferative dependent expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/uracil DNA glycosylase (UDG) gene and the induction of uracil DNA glycosylase activity was examined in human cells. Three different cell types were studied to determine whether the growth-dependent regulation of this multifunctional gene was a common characteristic of human cells. These included WI-38 normal embryonic lung fibroblasts, a Japanese Bloom's syndrome non-transformed skin fibroblast cell strain (GM-05289) and a lymphoblastoid cell line transformed by the Epstein-Barr virus. The Japanese Bloom's syndrome cells displayed the altered immunoreactivity with marker monoclonal antibody 40.10.09 which characterizes cells from this human genetic disorder. In noncycling human cells Northern blot analysis using a plasmid (pChug 20.1) which contained the human GAPDH/UDG cDNA revealed a single 1.6 kb transcript. In each case, the expression of this gene was increased during cell proliferation. This increase in GAPDH/UDG gene expression was identical to that observed for UDG enzyme activity. Further, using anti-human UDG monoclonal antibodies, there was a growth-dependent rise in immunoreactivity suggesting an increase in the level of antigenic protein. These results demonstrate that: (i) the expression of the GAPDH/UDG gene was dependent on the proliferative state of the cell; and (ii) a correlation existed between the transcription of this gene and the level of uracil DNA glycosylase enzyme activity.


Lack of correlation between DNA methylation and hepatocarcinogenesis in rats and hamsters treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Previous studies have demonstrated that the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced liver tumors in F344 rats but not in Syrian golden hamsters. The aim of this study was to determine whether there was a correlation between the persistence of O6-methylguanine (O6-mGua) adducts and the rate of recovery of O6-methylguanine-DNA methyltransferase (O6-mGuaT) after depletion in the liver and susceptibility to NNK in F344 rat and Syrian golden hamster injected s.c. with NNK (80 mg/kg). The levels of both 7-methylguanine and O6-mGua reached a maximum 24 h after NNK treatment. O6-mGua in NNK-treated rat liver was undetectable after 48 h. In the rat, the depletion of O6-mGuaT activity occurred within 4 h following NNK treatment. A subsequent rapid recovery of enzyme activity was observed 36 h after NNK exposure. In contrast, high levels of O6-mGua persisted in hamster liver DNA and no O6-mGuaT activity was detected up to 336 h after NNK injection. Thus, the persistence of O6-mGua in hamster liver is most likely related to a lack of recovery of the O6-mGuaT. These results suggested that factors other than O6-mGua may be determining NNK-induced hepatocarcinogenesis in rats. An aldehyde generated by alpha-hydroxylation of NNK, 4-oxo-4-(3-pyridyl)butanal, inhibited O6-mGuaT activity in rat hepatocytes, suggesting that this aldehyde contributes to the carcinogenicity of NNK by inhibiting this repair enzyme.


Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen, by rabbit nasal microsomes and cytochrome P450s NMa and NMb. Rabbit nasal olfactory and respiratory microsomes were found to catalyze the alpha-hydroxylation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with specific activities of 262 and 136 pmol/min/mg protein in the formation of keto aldehyde, and of 318 and 190 pmol/min/mg protein in the formation of keto alcohol respectively. The formation of NNK-N-oxide was observed in experiments with rabbit olfactory and respiratory microsomes, but not with rat nasal microsomes. However, the rat nasal microsomes had higher activity in catalyzing the alpha-hydroxylation of NNK. In a reconstituted system, rabbit P450NMa, a major constitutive P450 isozyme in nasal microsomes, displayed high activities in the formation of the keto aldehyde and the keto alcohol with apparent Km values of 15 and 9 microM respectively. In comparison, rabbit olfactory specific P450NMb had a low activity in catalyzing the formation of keto aldehyde (Km = 186 microM) and no activity in the formation of keto alcohol. The P450NMa-catalyzed oxidation of NNK was inhibited by nicotine and diallyl sulfide. Kinetic studies indicated that nicotine is a competitive inhibitor. These results demonstrate that enzymes in rabbit nasal microsomes, especially P450NMa, efficiently catalyze the bioactivation of NNK.


Activity of diverse tumor promoters in a keratinocyte co-culture model of initiated epidermis. The suppression of focal growth of initiated mouse keratinocytes by co-culture with normal keratinocytes can be overcome by treatment of co-cultures with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). This keratinocyte co-culture model was developed as an analog of initiated mouse epidermis to facilitate the study of tumor promotion in cell culture. A number of promoters of TPA-type [those with protein kinase C (PKC) as receptor] were compared to non-TPA-type promoters for activity in the keratinocyte co-culture model. The TPA-type promoters teleocidin and aplysiatoxin show comparable activity to that of TPA. Exposure of co-cultures to oleoyl-2-acetylglycerol, a diacylglycerol activator of PKC, also induces focal outgrowth of initiated cells, suggesting that PKC is likely to be involved in the mechanism of action of these compounds. However, the involvement of alternative pathways (not involving PKC directly) for clonal selection are evident since the non-TPA-type promoters okadaic acid, staurosporine and thapsigargin are also active in the assay. Thus, the keratinocyte co-culture model differs from fibroblast models of normal and neoplastic co-cultures in which only TPA-type promoters are active. In further contrast to certain fibroblast co-cultures, TPA does not inhibit cell-cell communication under conditions that suppress focus formation. Taken together with previous data, we conclude that the keratinocyte co-culture model may have broad application for detecting skin tumor promoters, and may be useful for dissecting the mechanism by which normal epidermal cells suppress the growth of initiated cells.


DNA adduct dosimetry and DNA repair in rats and pigs given repeated doses of procarbazine under conditions of carcinogenicity and human cancer chemotherapy respectively. Procarbazine (PCZ), an antineoplastic agent that produces methylated bases in DNA after metabolic activation, has been implicated in the development of secondary cancers in patients treated for a primary neoplasm. The repair of the important promutagenic lesion, O6-methylguanine (O6-meG) by O6-alkylguanine-DNA alkyl transferase (AGT) is believed to be crucial for the stability of O6-meG and for the tumorigenic outcome after exposure to methylating carcinogens. Using two different animal models, we investigated methyl DNA adduct dosimetry and DNA repair in (i) female rats given repeated doses of PCZ for 20 weeks under conditions of carcinogenicity, and (ii) female pigs administered repeated doses of PCZ for 4 weeks according to a regimen comparable to that given to human subjects undergoing cancer chemotherapy. After each successive week, four rats and three pigs were killed and tissues including blood, liver, mammary gland, spleen, thymus and lymph node were taken. The levels of O6-meG, 7-methylguanine (7-meG) in DNA and of AGT were determined in these tissues. In the rat, O6-meG in the liver DNA, and to a lesser degree in the spleen was efficiently removed throughout the 20 week dosing period, as indicated by the O6-meG/7-meG ratio being much less than 0.11. In the target organs, accumulation of O6-meG began in the mammary gland after 9 weeks, and in the lymph node and thymus after 3 weeks of dosing. Interestingly, the accumulation of O6-meG in the mammary gland correlates well with a concomitant decrease in AGT level as from week 10 and may be related to the induction of mammary gland tumors, first detected in two animals at week 10. In pigs, after a total dose of 750-4000 mg of PCZ, the range of 7-meG detected in leukocyte DNA was 21-66 mumol/mol G, which compares well with recent findings in cancer patients treated with PCZ. Similar levels of 7-meG were detected in the pig liver, thymus and lymph node. O6-MeG was only detectable in leukocyte DNA at week 4 with our present method. Compared with control pig tissues, a depressed AGT level was found in the leukocyte, lymph node and brain of the treated animals.


The influence of ventral UVA exposure on subsequent tumorigenesis in mice by UVA or UVB irradiation. Exposure to ultraviolet-B radiation (UVB: 280-315 nm) can result in a decreased immune response. This immune suppression can be restricted to the exposed skin site (local immune suppression) but may also be systemic. To investigate whether ultraviolet-A radiation (UVA: 315-400 nm) could also exert such a systemic effect, we performed the present investigation. The study consisted of two parts. Experiment I: 24 albino hairless mice (SKH:HRI) were ventrally exposed to UVA radiation for 300 days (glass-filtered Philips TLK09 fluorescent tubes, daily dose: 350 kJ/m2), while 24 control mice were left unexposed. After this period the control animals were still tumour free, but 60% of the exposed animals had developed abdominal tumours. Subsequently ventral exposures were stopped and both groups were dorsally exposed to identical UVB regimens (Westinghouse FS40, daily dose: 900 J/m2). Experiment II: this was virtually the same as experiment I, but here the mice were dorsally exposed to UVA radiation (glass-filtered Philips TLK09, daily dose: 290 kJ/m2) instead of UVB radiation. If we look at all tumours induced dorsally, we find no significant influence of pre-exposures to UVA radiation. This holds for dorsal UVB as well as for dorsal UVA exposures. In contrast to UVB, however, the UVA radiation induced many papillomas. Excluding the papillomas from the analysis we find that the induction of non-papillomas (mainly squamous cell carcinomas) under dorsal UVA exposure, is slightly enhanced in the ventrally pre-exposed group (difference significant at the P < 0.05 level). This suggests that UVA radiation induced only a weak systemic effect. Ventral UVA pre-exposure did not appear to affect dorsal skin irritation as expressed by scratch marks. The induction period for hyperkeratosis, however, was significantly shortened by the ventral UVA pre-exposure; this applied to dorsal UVB as well as dorsal UVA exposures.