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S A Belinsky

Publications and source records attributed to S A Belinsky.

At least 55 records · Page 3Linked to original sources
PubMed1993

Analysis of activated protooncogenes in B6C3F1 mouse liver tumors induced by ciprofibrate, a potent peroxisome proliferator.

Liver tumors from B6C3F1 mice induced by the potent peroxisome proliferator ciprofibrate, a hypolipidemic drug, were evaluated for the presence of transforming genes by the nude mouse tumorigenicity assay. As reported earlier, the tumors were not activated by a point mutation in codon 61 of H-ras. Two of the eight tumors examined contained a mutation in codon 13 or an H-ras gene mutated in codon 117. Screening of another 23 ciprofibrate-induced liver tumors by oligonucleotide hybridization analysis and direct DNA sequencing resulted in the identification of three tumor DNA samples with point mutations in codon 117 of the H-ras gene. In addition, another tumor sample contained a K-ras gene with a mutation in codon 61. Mutations in these codons have been seen only rarely in chemically induced liver tumors from this mouse strain. Of 15 spontaneous B6C3F1 liver tumors screened in the same manner, one exhibited a K-ras gene activated by a mutation in codon 13 and a second contained an H-ras gene activated by a mutation in codon 117. These ras gene mutations have not been reported previously from spontaneous liver tumors. The frequency and spectrum of ras oncogene mutations characterized in ciprofibrate-induced liver tumors differ significantly from the frequency and pattern identified in spontaneously occurring liver tumors. The results of this study with a limited number of samples suggest that ras protooncogene activation or activation of other protooncogenes that can be detected by the nude mouse tumorigenicity assay are not frequent events in the mechanism of carcinogenicity of the peroxisome proliferator ciprofibrate. However, the lower frequency and distinct pattern of H-ras mutations observed in these tumors disprove the assumption of promotion of spontaneous hepatocarcinogenesis by ciprofibrate.

3T3 Cells↗
PubMed1993

Metabolism and macromolecular interaction of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in cultured explants and epithelial cells of human buccal mucosa.

Metabolism and macromolecular interaction of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were studied in human buccal mucosa in vitro. Microautoradiographic analysis of [5-3H]NNK-exposed explant cultures demonstrated a uniform distribution of bound radioactivity in the mucosal epithelium, without significant binding in the underlying connective tissue. The metabolism of [5-3H]NNK at concentrations of both 6 and 100 microM resulted in seven identified metabolites in both explant and epithelial cell cultures. Formation of 4-(methylnitrosamino)-1-(3-pyridyl)butan-1-ol by carbonyl reduction of NNK accounted for almost 95% of the total metabolism, whereas the proportions of other metabolites obtained by alpha-carbon hydroxylation and pyridine N-oxidation reactions varied from undetectable levels to approximately 2% in both experimental systems. A positive correlation between concentration and the metabolic route associated with the formation of DNA methylating intermediates (alpha-hydroxylation at the N-methylene carbon) was found, i.e. when the concentration of NNK was raised from 6 to 100 microM, keto-acid formation which in part reflects DNA methylation was increased preferentially over ketoalcohol production, an index of DNA pyridyloxobutylation. Both the total rate of NNK metabolism and the amount of protein adducts were higher in cells from primary cultures up to the third passage than in explants cultured for 1 day. Between 10(-9) to 10(-4) M, neither NNK nor its precursor alkaloid nicotine affected the colony forming efficiency of normal and tumorous buccal epithelial cells, although at 10(-3) M each agent inhibited this function. Taken together, the results demonstrate the capability of human buccal mucosal epithelium to metabolize NNK by three major pathways, including those involved in the formation of adducts with cellular macromolecules.

Autoradiography↗
PubMed1993

Metabolism of nitrofluoranthenes by rat lung subcellular fractions.

The nitrofluoranthene (NF) family of compounds includes the potent pulmonary carcinogen 3,9-dinitrofluoranthene (3,9-DNF) and the weak carcinogen 3-nitrofluoranthene (3-NF). Although the specific molecular mechanisms involved in this difference in sensitivity for the induction of lung tumors in rats by 3,9-DNF and 3-NF have not been defined, these compounds most likely induce carcinogenesis by metabolic activation to electrophilic metabolites that bind DNA. The purpose of these investigations was to determine the activation pathways in the rat lung for the metabolism of the di-(3,9-DNF) and mono-nitroisomers (3-NF, 8-NF, 2-NF) of NFs. The metabolic rates of NFs were compared for lung subcellular fractions of pristine rats as well as rats previously treated with 3-methylcholanthrene (3-MC) or phenobarbital at levels that would induce cytochrome P450 enzymes. One major metabolite, the amino derivative, was detected by high pressure liquid chromatography following anaerobic incubation of rat lung cytosol with 3-NF, 8-NF, 2-NF or 3,9-DNF. 3,9-DNF was metabolized to its amino derivative, aminonitrofluoranthene, at a higher rate than 3-NF, 8-NF or 2-NF. Pretreatment of the rats with 3-MC or phenobarbital did not affect the metabolic rates of cytosolic reduction. Both 3-NF and 3,9-DNF were metabolized anaerobically to their amino derivatives by microsomal reductas(s). 3,9-DNF was metabolized twice as fast as 3-NF. The formation of the aminonitrofluoranthene metabolite was increased approximately 2 times with microsomes from 3-MC-induced rats, but was unaffected by microsomes from phenobarbital-treated rats. This suggests that the cytochrome P450 isozymes and reductase, which are induced by 3-MC, may be involved in the metabolism of 3-NF and 3,9-DNF. The metabolic products of 3-NF, formed aerobically, consisted of one major and three minor compounds. The major metabolite, tentatively identified as 3-NF-8-ol, was increased approximately 6 times using microsomes from 3-MC-induced rats. In contrast, 3,9-DNF metabolism was not detected aerobically with lung microsomes. Thus, ring hydroxylation was inhibited in the metabolism of 3,9-DNF, and the major pathway was nitroreduction. This higher rate of anaerobic metabolism of 3,9-DNF over 3-NF and the expected high reactivity of the putative N-acetoxy derivative formed from 3,9-DNF may be responsible for the differential potency for lung cancer induction by these two carcinogens.

Aerobiosis↗
PubMed1992

Role of the alveolar type II cell in the development and progression of pulmonary tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the A/J mouse.

The role of the type II cell in the development of pulmonary tumors induced in the adult A/J mouse (6 weeks of age) by treatment with a single dose (100 mg/kg, i.p.) of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was investigated. Twenty-four h following treatment with NNK, the concentration of O6-methylguanine was similar in Clara and type II cells. However, hyperplasias were detected only along the alveolar septa in lungs 14 weeks after carcinogen treatment. Examination of the ultrastructure of several hyperplasias revealed that the proliferating cells resembled type II pneumocytes. The proliferating cells were cuboidal in shape, with centrally localized ovoid nuclei characterized by minor indentations. Lamellar bodies, one of the major hallmarks of the type II cell, were present in the cytoplasm. The progression of pulmonary lesions was followed by sacrificing mice at 4-week intervals from 14 to 54 weeks after treatment with NNK. From 34 to 42 weeks after treatment, progression to neoplasia was demonstrated by a decline in the frequency of hyperplasias and an increase in the frequency of adenomas. Approximately 50% of the adenomas were observed arising within hyperplasias. Carcinomas appeared to increase in frequency 34 weeks after carcinogen treatment and comprised greater than 50% of the pulmonary lesions by 54 weeks. Approximately 30% of the carcinomas were observed arising within adenomas. The growth pattern of carcinomas began to change from solid to mixed (solid and papillary) 42 weeks after NNK. Moreover, electron micrographic analysis demonstrated that, within a hyperplasia, proliferating type II cells could change from cuboidal to columnar in shape and could also exhibit nuclear indentations, both characteristics displayed by the Clara cell. Thus, this divergence of the type II cell from its well characterized morphological features indicates that the selective growth advantage which these initiated cells possess can result in changes to the normal ultrastructure of this cell as it progresses toward malignancy. DNA was isolated from 20 hyperplasias and screened for the presence of an activated K-ras gene. This gene was activated in 17 of 20 lesions, with 85% of the mutations involving a GC to AT transition within codon 12 (GGT to GAT), a mutation consistent with base mispairing produced by the formation of the O6-methylguanine adduct. This specificity for activation of the K-ras gene was identical to that observed previously in adenocarcinomas induced by NNK.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoma↗
PubMed1991

Effect of allyl alcohol on xanthine dehydrogenase activity in the perfused rat liver.

Xanthine oxidase has been implicated in the production of reactive oxygen species and cell injury produced by various toxic compounds. Since allyl alcohol injuries the liver by an oxygen-dependent mechanism, we examined the actions of this hepatotoxicant on the conversion of xanthine dehydrogenase into xanthine oxidase in perfused livers. A microassay for NAD(+)-dependent xanthine dehydrogenase, based on measuring the production of NADH fluorometrically under anaerobic conditions, was developed and used to examine the actions of allyl alcohol on this activity in periportal and pericentral regions of the liver lobule. The oxygen-dependent activity, xanthine oxidase, was monitored in whole liver homogenates by uric acid formation at 302 nm under aerobic conditions. Perfusion of the liver with allyl alcohol (350 microM) increased xanthine oxidase and decreased xanthine dehydrogenase in whole liver consistent with the hypothesis that allyl alcohol enhanced calcium-dependent proteolytic conversion of the NAD(+)-dependent to the O2-dependent form. Xanthine dehydrogenase was higher in pericentral than in periportal regions of the liver lobule and tended to decrease selectively in periportal zones of livers exposed to allyl alcohol. O2 uptake was stimulated transiently by allyl alcohol followed by subsequent inhibition of respiration. These results are consistent with the idea that conversion of NAD(+)-dependent xanthine dehydrogenase to xanthine oxidase is involved in the zone-specific hepatotoxicity of allyl alcohol.

1-Propanol↗
PubMed1991

Role of ras protooncogene activation in the formation of spontaneous and nitrosamine-induced lung tumors in the resistant C3H mouse.

The role of ras activation in the formation of spontaneous and chemically induced tumors was evaluated in the C3H mouse, a strain that has a low incidence of spontaneous lung tumors. Lung tumors were induced in C3H mice by treatment with 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 50 mg/kg, or nitrosodimethylamine (NDMA), 3 mg/kg for 7 weeks (3 times/week, i.p.). Eleven tumors from each treatment group were evaluated for activated ras genes by direct sequencing and oligonucleotide hybridization to slot blots of amplified DNA from these tumors. An activated K-ras gene was detected in 100% of NDMA- and NNK-induced lung tumors, and the activating mutation detected in all samples was a GC to AT transition (GGT to GAT) in codon 12. In contrast, only 40% of the seven spontaneous lung tumors analyzed contained an activated K-ras gene and the mutations identified were not localized to either a specific base or codon. Both NNK and NDMA can be activated via alpha-hydroxylation to methylating agents. The GC to AT mutation observed in codon 12 in the nitrosamine-induced tumors is consistent with the formation of an O6-methylguanine (O6MG) adduct. Similar concentrations (13-15 pmoles/mumol deoxyguanosine) of this promutagenic adduct were detected in lungs during treatment with either NNK or NDMA. Thus, both these nitrosamines appear to activate the K-ras gene in lung through a direct genotoxic mechanism involving the formation of the O6MG adduct. The frequency of K-ras activation was similar in chemically induced lung tumors from the sensitive A/J strain and the C3H mouse, indicating that susceptibility for neoplasia in these stains is not related to the ability to activate this gene. Although tumors were induced in lung from 100% of C3H mice following chronic carcinogen exposure, both the size and the multiplicity was significantly less, while latency was longer than that observed in the A/J mouse. These differences could not be attributed to an altered propensity for DNA damage, but rather suggest that genetic loci which regulate clonal expansion and growth of initiated cells play a major role in the susceptibility of pulmonary neoplasia.

Animals↗
PubMed1990

Dose-response relationship between O6-methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The relationship between the formation of O6-methylguanine (O6MG) and the induction of lung, liver, and nasal tumors in the Fisher 344 rat by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was examined in a dose-response study. Animals were treated for 20 wk (3 times/wk) with concentrations of NNK ranging from 0.03 to 50 mg/kg to induce tumors. Steady-state concentrations of O6MG were quantitated, and cytotoxicity was assessed in target cells and tissues after 4 wk of treatment with NNK. No cytotoxicity was detected in the lung during treatment with NNK. The formation of O6MG was greatest in Clara cells compared with macrophages, type II cells, small cells, and whole lung at all doses examined. The difference in adduct concentration between the Clara cell and other pulmonary cell types was most pronounced with low doses of carcinogen. The O6MG:dose ratio, an index of alkylation efficiency, increased 29-fold as the dose of NNK was decreased from 50 to 1 mg/kg of carcinogen. In contrast, only a small increase in alkylation efficiency was observed in type II cells and whole lung. A significant number of tumors were induced in the lung at doses of 0.1 to 50 mg/kg with incidences ranging from 10% at the lowest dose up to 87% in the group of animals which received 50 mg/kg of NNK. A linear relationship was observed when the concentration of O6MG in Clara cells as a function of dose was plotted against the corresponding tumor incidence. This relationship was not observed using DNA adduct concentrations in type II cells or whole lung. The development of pulmonary tumors appeared to involve the formation of alveolar hyperplasias which progressed to adenomas and finally to carcinomas. The majority of adenomas were solid, whereas carcinomas were mainly papillary. Examination of the ultrastructure of the hyperplasias, adenomas, and carcinomas revealed morphological structures (e.g., lamellar bodies, tubular myelin) which are associated with type II cells. Thus, these data suggest that the majority of neoplasms in the lung begin as type II cell proliferations with progression to adenomas and carcinomas within the areas of hyperplasia. The lack of agreement between biochemical and morphological findings makes it difficult to hypothesize a cell of origin for the pulmonary neoplasms. In contrast to the lung, tumors were induced in the liver and nasal passages only after exposure to high doses of NNK. Moreover, both the formation of DNA adducts and cytotoxicity appear obligatory for the generation of tumors in these tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Relationship between the formation of promutagenic adducts and the activation of the K-ras protooncogene in lung tumors from A/J mice treated with nitrosamines.

Lung and liver tumors were induced in female A/J mice after treatment for 7 weeks (3 times/week, i.p.) with either 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (50 mg/kg) or nitrosodimethylamine (NDMA) (3 mg/kg). Both compounds can be activated via alpha-hydroxylation to methylating agents, while NNK may also undergo hydroxylation at the N-methyl carbon to form a pyridyloxobutylated adduct. The purpose of these studies was to identify and characterize the activated oncogenes present in tumors induced by NDMA and NNK. Following transfection of high molecular weight DNA onto NIH/3T3 mouse fibroblasts, transforming genes were detected in 90% of both NNK- (10 of 11) and NDMA- (9 of 10) induced lung tumors. In contrast, transformation of NIH/3T3 fibroblasts was observed only in 40% (2 of 5) and 13% (1 of 8) of the liver tumors from NNK- and NDMA-treated mice, respectively. Southern blot analysis indicated that the transforming gene present in all lung tumors was an activated K-ras oncogene. Both rearranged bands and amplified signals were detected in the transfectants. The one transformant from the NDMA-induced liver tumor contained an activated K-ras gene. In contrast, the two liver transformants from NNK-induced tumors did not contain an activated ras or raf gene. Hybridization with oligonucleotide probes that were centered around either codon 12 or 61 of the K-ras gene were utilized to localize the mutations. Activation of this gene appeared to occur largely via a mutation in codon 12 (15 of 20 transformants) and was observed with a similar frequency in pulmonary tumors induced by either compound. The remaining mutations were found in codon 61. The specific mutation within these two codons was determined by amplifying the exon containing the base change, followed by direct sequencing. With one exception the mutation observed in codon 12 was a GC to AT transition (GGT to GAT). One transformant contained a GC to TA transversion. The activating mutation detected in codon 61 was always an AT to GC transition of the middle A (CAA to CGA). The GC to AT mutation observed in codon 12 is consistent with the formation of the O6-methylguanine adduct. Similar concentrations (23 to 32 pmol/mumol deoxyguanosine) of this promutagenic adduct were detected in lungs during treatment with either NNK or NDMA.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗
PubMed1989

Cell specificity for the pulmonary metabolism of tobacco-specific nitrosamines in the Fischer rat.

The activity and distribution of the metabolic pathways of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and the structurally related nitrosamine, N'-nitrosonornicotine (NNN) were examined in pulmonary cells from F344 rats in order to investigate the mechanisms by which NNK and NNAL, but not NNN, cause lung tumors. The tritium labeled nitrosamines were incubated with Clara cells, alveolar macrophages, alveolar type II cells, or small cells and metabolites were analyzed by HPLC. O6-Methyl-guanine (O6MG) formation was also quantified in the cells incubated with NNK. Clara cells metabolized all compounds more extensively than the other cell types. Total alpha-hydroxylation, carbonyl reduction to NNAL, and pyridine N-oxidation in cells incubated with NNK, as well as concentrations of O6MG in DNA were higher in Clara cells than in other cell types. Carbonyl reduction of NNK predominated over the other metabolic pathways in all cell types. The high activity for alpha-hydroxylation of NNK in Clara cells is consistent with previous studies which proposed that the cell specificity for O6MG formation and the accumulation of this adduct during low-dose exposure to NNK may stem from the presence of a high affinity pathway in Clara cells for NNK activation. Metabolism of NNAL by alpha-hydroxylation, and by reconversion to NNK followed by alpha-hydroxylation were observed. Total alpha-hydroxylation of NNAL was less extensive than alpha-hydroxylation of NNK. NNN was metabolized by both the 2'- and 5'-alpha-hydroxylation pathways. 2'-Hydroxylation of NNN produces the same DNA pyridyloxobutylating agent as does methyl hydroxylation of NNK. However, NNN is not a methylating agent and does not induce lung tumors in rats. Metabolism of NNN by 2'-hydroxylation was, depending on cell type, 41-85% as extensive as total alpha-hydroxylation of NNK, indicating that the rates of formation of the DNA pyridyloxobutylating agent were similar from NNN and NNK. The results of this study demonstrate that Clara cells have a high capacity to metabolically activate NNK, NNAL and NNN and provide further support for the hypothesis that DNA methylation of pulmonary cells is important in NNK carcinogenesis.

Alkylation↗
PubMed1989

Interactions between glycolysis and mixed function oxidation: studies with 7-ethoxycoumarin in perfused livers from beta-naphthoflavone-treated rats.

Interaction between glycolysis and mitochondrial oxidations to supply reducing equivalents at high rates for mixed function oxidation was evaluated in the perfused liver after treatment of rats with beta-naphthoflavone. Livers from fasted beta-naphthoflavone-treated rats were employed because rates of 7-ethoxycoumarin O-deethylation were constant (16 mumol/g/hr) for at least 1 hr of perfusion. Preinfusion with KCN, an inhibitor of oxidative phosphorylation, caused the rate of 7-ethoxycoumarin O-deethylation to decline by 60% over 30 min of perfusion. The decline in rates of mixed function oxidation in the intact liver was not due to a direct effect of KCN on cytochrome P-450, inasmuch as cyanide did not diminish rates of 7-ethoxycoumarin O-deethylation by isolated microsomes. Cyanide rapidly decreased hepatic oxygen uptake by 70% and increased rates of glycolysis (lactate plus pyruvate production) from less than 10 to over 60 mumol/g/hr. Rates of glycolysis and mixed function oxidation subsequently declined in parallel during infusion of KCN. Infusion of ethanol (20 mM), a known inhibitor of glycolysis, decreased the stimulation of glycolysis caused by KCN to 20 mumol/g/hr and lowered maximal rates of 7-hydroxycoumarin production to about 6 mumol/g/hr. Both mixed function oxidation and glycolysis also declined in parallel over 30 min of perfusion in the presence of ethanol and KCN. When cyanide infusion was terminated, rates of oxygen uptake returned rapidly to basal values; however, rates of mixed function oxidation remained low. In contrast, infusion of ethanol in the absence of cyanide had no effect on rates of mixed function oxidation. Infusion of glucose (30 mM) or pyruvate (1 mM) after KCN restored maximal rates of mixed function oxidation in parallel with increases in rates of glycolysis. In contrast to results obtained in livers from fasted rats, cyanide and ethanol had little effect on 7-ethoxycoumarin O-deethylation in livers from fed rats. Taken together, these results argue strongly that rates of mixed function oxidation in the intact livers of fasted rats are sustained by reducing equivalents derived from mitochondrial oxidations. Glycolysis can supply substrates needed for the transport of reducing equivalents from the mitochondria into the cytosol for mixed function oxidation. Because glycogen reserves are minimal in the fasted state, rates of glycolysis and mixed function oxidation declined in parallel during the infusion of cyanide, because reducing equivalents derived from mitochondria are not available.

Animals↗
PubMed1988

Factors regulating activation and DNA alkylation by 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and nitrosodimethylamine in rat lung and isolated lung cells, and the relationship to carcinogenicity.

The molecular dosimetry for O6-methylguanine (O6MG) formation in DNA from rat lung and pulmonary cells was compared following treatment for 4 days with equimolar doses of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen or nitrosodimethylamine (NDMA), a weak carcinogen in rat lung. The dose response for O6MG formation from NNK was biphasic; the O6MG to dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen was decreased. In contrast, the dose-response curve for methylation by NDMA appeared opposite of that for NNK with alkylation efficiency increasing as a function of dose. These results suggested that high and low Km pathways exist for the activation of NNK, whereas only high Km pathways may be involved in NDMA activation. Furthermore, DNA methylation by NNK was cell selective with the highest levels in the Clara cell, whereas methylation by NDMA was not. DNA methylation in the Clara cell was 50-fold greater by NNK than by NDMA at equimolar doses (0.005 mmol/kg). Thus, differences in O6MG formation, specifically the presence of a high affinity pathway in the Clara cell for activation of NNK, may explain why following low dose exposure, NNK is a potent pulmonary carcinogen while NDMA is not. Different cytochrome P-450 isozymes also appear to be involved in the activation of NNK and NDMA. Inhibition of in vitro methylation (with calf thymus DNA and lung microsomes) by antibodies to cytochrome P-450 isozymes provided evidence that a homolog of rabbit cytochrome P-450(2) (cytochrome P-450b) may be important in the activation of NNK in rat lung, whereas cytochrome P-450(5) may activate NDMA. A 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible cytochrome P-450 isozyme (P-450c) may also be involved in the activation of NNK but not NDMA. Treatment with TCDD increased both NNK activation by pulmonary microsomes and the formation of O6MG in Clara cells and type II cells incubated in vitro with NNK. alpha-Naphthoflavone (alpha-NF), a specific inhibitor of cytochrome P-450c reversed the increase in methylation by TCDD-induced microsomes but did not inhibit in vitro activation of NNK using microsomes from untreated rats. However, NNK mediated O6MG formation in Clara cells, but not in type II cells incubated with alpha-NF, was decreased by 21%. These data indicate that both cytochrome P-450b and P-450c are probably involved in the activation of NNK in Clara cells from untreated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylation↗
PubMed1988

Cell specific differences in O6-methylguanine-DNA methyltransferase activity and removal of O6-methylguanine in rat pulmonary cells.

Previous studies have demonstrated that cell specificity exists for the alkylation of DNA from lung cells following treatment of rats with the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The concentration of the promutagenic adduct O6-methylguanine (O6MG) was found to be greatest in Clara cells followed by macrophages, type II cells and alveolar small cells. The purpose of this study was to measure the activity of the repair protein O6-methylguanine-DNA methyltransferase (O6MGMT) and to determine whether differences exist for the removal of O6MG among pulmonary cell types. Constitutive activity of O6MGMT was 2-fold greater in macrophages and type II cells than alveolar small cells and Clara cells. Treatment for 4 days with NNK (10 mg/kg/day) had no effect on O6MGMT activity in macrophages, but decreased activity in alveolar small cells and type II cells by 57 and 84%, respectively. O6MGMT activity was reduced to below limits of detection in Clara cells following treatment with NNK. The effect of NNK on O6MGMT activity was consistent with rates of removal of O6MG in macrophages and Clara cells. The loss of O6MG from DNA of macrophages followed first order kinetics (t1/2 = 48 h) while very little loss of this adduct was observed in Clara cells over an 8 day period following cessation of carcinogen treatment. Even though O6MGMT activity was reduced in alveolar small cells and type II cells, approximately 90% of the O6MG bound to DNA in these cell types was removed within 8 days after treatment was discontinued. The loss of O6MG from pulmonary cells appears to result largely from the removal of this adduct by O6MGMT since rates of cell turnover were very low (0.5-1.5%/day) in the lung and were not affected by treatment with NNK. This study indicates that the activity of O6MGMT and the rate of resynthesis of this repair enzyme differ considerably among pulmonary cells following the methylation of DNA. The high concentration of O6MG in Clara cells and the low rate of repair of this promutagenic adduct may be critical factors in the potent pulmonary carcinogenicity induced by the tobacco specific carcinogen NNK.

Animals↗
PubMed1987

Molecular dosimetry of DNA adduct formation and cell toxicity in rat nasal mucosa following exposure to the tobacco specific nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and their relationship to induction of neoplasia.

The molecular dosimetry of O6-methylguanine (O6MG) formation in DNA and cytotoxicity in respiratory and olfactory mucosa was determined during administration of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to male Fischer 344 rats. The dose response for O6MG formation differed considerably between respiratory and olfactory mucosa. The dose response was nonlinear in respiratory mucosa where the slope of the curve was very large for doses of NNK ranging from 0.3 to 3.0 mg/kg but much smaller in the dose range of 10 to 100 mg/kg. In contract, the dose response in the olfactory mucosa did not demonstrate such a large change in slope over the same dose range. The concentration of O6MG formed to dose of NNK ratio, an index of efficiency of alkylation, increased dramatically only in the respiratory mucosa as the dose of NNK was decreased from 100 to 0.3 mg/kg. The concentration of O6MG was four times greater in respiratory than olfactory mucosa after treatment of rats with 1 mg/kg NNK. Alkylation in the two regions of the nose became similar as the dose of NNK was increased. In rats treated for up to 12 days with NNK (10 mg/kg/day), the concentration of O6MG was 60 to 90% greater in respiratory than olfactory mucosa throughout treatment. Regional differences in the amount of O6MG formed may stem from the presence of a low Km pathway for biotransformation of NNK in the cells of the respiratory mucosa. This conclusion is supported by autoradiographic studies. Four h after treatment with 1 mg/kg [3H]NNK, silver grains were more heavily concentrated in respiratory than olfactory epithelium. Histopathological examination of the nasal passages revealed dose related, cell specific differences in toxicity following treatment of rats with 10, 30, or 100 mg/kg NNK for 12 days. No toxicity was observed in the nose when 1 mg/kg NNK was administered. Bowman's glands underlying the olfactory mucosa and Steno's glands were the most sensitive sites for toxicity, exhibiting necrosis after as little as 2 days of treatment with 10 mg/kg NNK. Damage to these glands progressed in a dose- and time-dependent manner. Respiratory epithelium exhibited only mild toxicity while basal cell metaplasia was evident in olfactory epithelium. Rats treated with NNK for 20 weeks (50 mg/kg, three times a week) had a 45% incidence of carcinomas in the olfactory region. These neoplasms appeared to arise from Bowman's glands. In contrast, there was only a 5% incidence of malignant neoplasia and a 29% incidence of benign neoplasia in the respiratory region.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Cell selective alkylation of DNA in rat lung following low dose exposure to the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone.

The molecular dosimetry of O6-methylguanine (O6MG) in DNA from lung and specific cell populations isolated from lung was determined during multiple administrations of the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to Fischer 344 rats. O6MG accumulated with doses of NNK ranging from 0.1 to 100 mg/kg/day. The dose response for NNK was nonlinear; the O6MG to dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen decreased. These data suggest that low and high Km pathways may exist for activation of NNK to a methylating agent. Marked differences in O6MG concentration were observed in specific lung cell populations. The Clara cell, one of the suggested progenitor cells for nitrosamine-induced neoplasia, was found to possess the greatest concentration of O6MG. Moreover, as the dose of NNK was decreased from 100 to 0.3 mg/kg, the alkylation efficiency in this cell population increased 38-fold. The high level of DNA adduct formation in Clara cells following low dose exposure to NNK was supported by autoradiographic studies. Four h after treatment with 1 mg/kg [3H]NNK, silver grains were more heavily concentrated over Clara cells than over other cell types in the lung. Comparative studies on dimethylnitrosamine, a weak carcinogen in the rat lung, did not demonstrate this cell specificity for DNA alkylation. Thus, the presence of a high affinity pathway in the Clara cell for activation of NNK may contribute to the carcinogenicity of this tobacco specific carcinogen.

Alkylation↗
PubMed1987

DNA adducts as a dosimeter for risk estimation.

The dose response for O6-methylguanine (O6MG) formation and cytotoxicity was determined in lung and nasal mucosa from Fischer 344 rats during multiple dose administration of the tobacco-specific nitrosamine-4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). O6MG accumulated in the lung following treatment for 12 days with doses of NNK from 0.3 to 100 mg/kg/day. The dose response for NNK was nonlinear; the O6MG-to-dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen decreased. These data suggest that low- and high-Km pathways may exist for activation of NNK to a methylating agent. Marked differences in O6MG concentration were observed in specific lung cell populations. The Clara cell, one of the suggested progenitor cells for nitrosamine-induced neoplasia, was found to possess the greatest concentration of O6MG. Moreover, as the dose of NNK was decreased from 100 to 0.3 mg/kg, the alkylation efficiency in this cell population increased 38-fold. The presence of a high-affinity pathway in the Clara cell for activation of NNK may contribute to the potent carcinogenicity observed following low-dose exposure to this tobacco-specific carcinogen. The dose response for O6MG formation differed considerably between the respiratory and olfactory mucosa from the nasal passages of the rat. The dose response was nonlinear in respiratory mucosa but linear in olfactory mucosa. The alkylation efficiency increased dramatically only in the respiratory mucosa as the dose of NNK was decreased. These studies suggest that a low Km pathway for NNK activation is also present in the nose and that this pathway is localized predominantly in the respiratory region.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1987

High- to low-dose extrapolation: critical determinants involved in the dose response of carcinogenic substances.

Recent investigations on mechanism of carcinogenesis have demonstrated important quantitative relationships between the induction of neoplasia, the molecular dose of promutagenic DNA adducts and their efficiency for causing base-pair mismatch, and the extent of cell proliferation in target organ. These factors are involved in the multistage process of carcinogenesis, including initiation, promotion, and progression. The molecular dose of DNA adducts can exhibit supralinear, linear, or sublinear relationships to external dose due to differences in absorption, biotransformation, and DNA repair at high versus low doses. In contrast, increased cell proliferation is a common phenomena that is associated with exposures to relatively high doses of toxic chemicals. As such, it enhances the carcinogenic response at high doses, but has little effect at low doses. Since data on cell proliferation can be obtained for any exposure scenario and molecular dosimetry studies are beginning to emerge on selected chemical carcinogens, methods are needed so that these critical factors can be utilized in extrapolation from high to low doses and across species. The use of such information may provide a scientific basis for quantitative risk assessment.

Animals↗
PubMed1987

Effect of beta-naphthoflavone on mitochondrial supply of reducing equivalents for monooxygenation in periportal and pericentral regions of the liver lobule.

Rates of 7-ethoxycoumarin O-deethylation were determined in periportal and pericentral regions of the liver lobule in livers from corn oil- and beta-naphthoflavone-treated rats by monitoring the conversion of nonfluorescent 7-ethoxycoumarin to fluorescent 7-hydroxycoumarin with micro-light guides. Rates of monooxygenation in livers from fed, corn oil-treated rats of 1.4 mumol/g/hr were increased markedly to around 21 mumol/g/hr in both regions of the liver lobule after treatment of rats with beta-naphthoflavone. Fasting or treatment with 6-aminonicotinamide diminished the generation of NADPH by the pentose cycle, whereas KCN decreased NADPH generation via mitochondria. Fasting and 6-aminonicotinamide treatment decreased monooxygenation about 0.5 mumol/g/hr in both regions of the liver lobule in livers from corn oil-treated rats and around 5 mumol/g/hr in livers from beta-naphthoflavone-treated rats. KCN decreased rates about 0.5 mumol/g/hr in both regions of the lobule in livers from fed, corn oil-treated rats and nearly completely in livers from fasted rats. Rates declined from 14 to less than 2 mumol/g/hr in livers from fasted, beta-naphthoflavone-treated rats following 30-40 min of perfusion with cyanide. These data indicate that mitochondrial oxidations are the predominant source of reducing equivalents for monooxygenation in both regions of the liver lobule in livers from beta-naphthoflavone-treated rats. Activation of urea synthesis by infusion of ammonia, a process requiring mitochondrial NADPH, inhibited the metabolism of 7-ethoxycoumarin by 30%. Malate, which is a substrate for the malic enzyme shuttle mechanism involved in the transfer of reducing equivalents from the mitochondria to the cytosol, increased 10-fold during infusion of 7-ethoxycoumarin in livers from beta-naphthoflavone-treated rats but less than 3-fold in livers from control rats. Taken together, these data indicate that high rates of 7-hydroxycoumarin production in livers from beta-naphthoflavone-treated rats are sustained by increased rates of NADPH generation from mitochondrial sources.

6-Aminonicotinamide↗