Biomedical subjects

A Castonguay

Publications and source records attributed to A Castonguay.

At least 55 records · Page 3Linked to original sources

PubMed1993

Dose-dependent ras mutation spectra in N-nitrosodiethylamine induced mouse liver tumors and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced mouse lung tumors.

In a previous study, the spectrum of H-ras mutations detected in B6C3F1 mouse liver tumors induced by 5, 50 or 150 mumol/kg body wt of N-nitrosodiethylamine (NDEA) was similar to that in spontaneous B6C3F1 mouse liver tumors, suggesting that activation of the H-ras gene in NDEA-induced mouse liver tumors may not be the direct result of the chemical interaction with the H-ras gene. In the present study, mutations in the H-ras oncogene from B6C3F1 mouse liver tumors induced by 5 or 50 mumol/kg body wt of NDEA were characterized by DNA amplification with polymerase chain reaction (PCR), single-strand conformation of polymorphism (SSCP) and direct sequence analysis. Twenty-one of 66 NDEA-induced B6C3F1 mouse liver tumors contained activated H-ras gene with 2 of 21 having a CG to AT transversion at the first base of codon 61, 17 of 21 having AT to GC transition and 2 of 21 having an AT to TA transversion at the second base of codon 61 in the H-ras gene. The predominant mutation, AT to GC transition (17/21, 81%) is consistent with the formation of O4-ethylthymine adduct, and is distinct from the predominant CG to AT transversion (50%) at the first base of codon 61 detected in H-ras gene from NDEA-induced B6C3F1 mouse liver tumors in a previous study by Stowers et al. Mutations in the K-ras oncogene from 59 A/J mouse lung tumors induced by 0.53 mmol/kg body wt of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were also characterized by using the above mentioned methods. Forty-six of 59 NNK-induced A/J mouse lung tumors contained activated K-ras genes. All 46 (100%) of the activated K-ras gene had GC to AT transitions at the second base of codon 12. The same mutation was observed in 70% (7/10) of the K-ras oncogene from A/J lung tumors induced by 4.8 mmol/kg body wt (given in 21 doses) of NNK. These data suggest that other factors in addition to genotoxic effect might be involved in the induction of rodent tumors by some carcinogens when given at higher doses. Therefore, further studies to compare the dose-dependent differences in the profile of ras mutations induced by chemical carcinogens may help to assess human cancer risk. Mutation(s) in exons 5-8 of the p53 gene was not found in these NDEA-induced mouse liver tumors and NNK-induced mouse lung tumors.

Animals↗

PubMed1993

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human kidney epithelial cells transfected with rat CYP2B1 cDNA.

In all species where it has been tested, the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been shown to be a potent carcinogen, and NNK and other nitrosamines may play a role in human tobacco-related carcinogenesis. Purified rat CYP2B1 has been shown to metabolize NNK, and the CYP2B1 gene is expressed constitutively in rat lung. The objectives of this study were to test the capacity of CYP2B1, synthesized from a rat hepatic cDNA in Ad293 cells, to metabolize NNK, and to define the type and the proportions of the final metabolites produced. Ad293 cells were transfected with a CYP2B1 expression vector (pMT2-2B1), or with a control vector and incubated in culture medium containing [3H]NNK, after which alpha-carbon hydroxylation and pyridine N-oxidation metabolites were identified by HPLC analysis and quantitated by scintillation counting. pMT2-2B1-transfected cells were capable of catalyzing alpha-carbon hydroxylation and pyridine N-oxidation of NNK, although the reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol(NNAL) was the major metabolite formed in cells regardless of transfection treatment. The total amount of alpha-carbon hydroxylation metabolites produced by pMT2-2B1-transfected cells was greater than that of pyridine N-oxidation metabolites. However, pMT2-2B1 transfected cells produced approximately ten-fold more pyridine N-oxidation metabolites and only two-fold more alpha-carbon hydroxylation metabolites than control cells. Furthermore, the amount of NNAL-N-oxide was much lower than that of NNK-N-oxide in the medium of pMT2-2B1-transfected cells, even though the amount of available NNAL, resulting from carbonyl reduction of NNK, was very high; this suggests that NNAL is poorly N-oxidized by CYP2B1 compared to NNK. These results show that within living cells NNK was metabolized by CYP2B1 via both the pyridine N-oxidation and alpha-carbon hydroxylation pathways. However, CYP2B1 preferentially catalyzed pyridine N-oxidation, which is considered to be a deactivation reaction.

Animals↗

PubMed1993

32P-postlabelling analysis of DNA-adducts formed by 4-(N-carbethoxy-N-nitrosamino)-(3-pyridyl)-1-butanone and 4-oxo-4-(3-pyridyl)- butanal.

The tobacco-specific nitrosamine, 4-(N-nitrosomethylamino)-1-(3-pyridyl)- 1-butanone (NNK) can be activated to DNA-binding species by two pathways: methylene hydroxylation leads to an equimolar formation of methyldiazohydroxide and 4-oxo-4-(3-pyridyl)-butanal followed by DNA methylation, while methyl hydroxylation leads to DNA pyridyloxobutylation via the 4-(3-pyridyl)-4-oxobutyldiazohydroxide (Hecht et al., 1988; Spratt et al., 1989). We report here that the ketoaldehyde, 4-oxo-4-(3-pyridyl)-butanal, as well as 4-(N-carbethoxy-N-nitrosamino)-1-(3- pyridyl)-1-butanone (NNC), a model compound for pyridyloxobutylation, form DNA adducts in vitro, as detected by 32P-postlabelling assays. The two major adduct spots of both compounds were chromatographically indistinguishable, suggesting structurally similar DNA adducts.

Animals↗

PubMed1992

Antimutagenic effects of polyphenolic compounds.

Smokers expose themselves to potent carcinogens daily. One of them is the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Since estimates are that humans consume 1 g of phenolic compounds/day, we investigated the inhibitory effects of five structurally related polyphenolic compounds on the mutagenicity of NNK in Salmonella typhimurium TA1535. NNK at a concentration of 80 mM was activated by hamster liver microsomes. The antimutagenic efficacies were dose-related between the non-toxic concentrations of 0.1 and 0.5 mmol/dish in the following order: esculetin > ellagic acid > (+)-catechin > propyl gallate > (-)esculin. At the highest non-toxic dose tested (0.5 mmol/dish), these polyphenolics inhibited mutagenesis in TA1535 by 77%, 67%, 62%, 59% and 53%, respectively. The results of this study demonstrated that polyphenolic compounds may inhibit the activation of NNK.

Animals↗

PubMed1992

Effects of NSAIDs on NNK-induced pulmonary and gastric tumorigenesis in A/J mice.

Non-steroidal anti-inflammatory drugs (NS-AIDs) are among the most widely prescribed drugs. In this study, we compared the efficacies of four NSAIDs to inhibit lung tumorigenesis in A/J mice. The tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was given in drinking water between week 0 and week +7. Groups of 25 mice were fed sulindac (123 mg/kg diet), ibuprofen (263 mg/kg), piroxicam (25 mg/kg) or naproxen (230 mg/kg) in AIN-76A diet from week -2 to the end of the bioassay (week +23). Sulindac was the most effective inhibitor and reduced lung tumor multiplicity by 51%. Ibuprofen and piroxicam reduced lung multiplicity by 38% and 30%, respectively. Naproxen demonstrated no inhibitory capacity. Forestomach tumor multiplicity and incidence were both reduced by sulindac and ibuprofen. Sulindac administered from week -2 to week +7 was less effective (28% inhibition) than when given throughout the bioassay. Sulindac induced more intestinal adhesions than any other NSAID and was directly related to the cumulative dose of sulindac. These results show that chemoprevention of lung tumorigenesis by NSAIDs is not limited to sulindac although it is the most effective.

Animals↗

PubMed1992

Expression of the cytochrome P4502E and 2B gene families in the lungs and livers of nonpregnant, pregnant, and fetal hamsters.

Members of the cytochrome P4502E and 2B gene families have been implicated in the activation of nitrosamines to reactive species capable of binding to cellular macromolecules and initiating tumor formation in various rodent species. This study was initiated to determine the relative prevalence of these isozymes and their response to ethanol during pregnancy and late gestation. Nonpregnant and pregnant hamsters were given a 10% ethanol solution in their drinking water for 10 days (gestation days 5-15) prior to being killed. RNA blot analysis of liver and lung tissue from nonpregnant, pregnant, and fetal hamsters demonstrated tissue-specific expression of CYP2E and 2B in adult and fetal animals. The levels of RNA expression of both P450s in fetal hamsters were less than 30% of nonpregnant adult values. In pregnant hamsters, the hepatic levels of CYP2E and 2B RNAs were decreased compared to nonpregnant animals. In contrast, the pulmonary levels of CYP2B RNA were increased in pregnant versus non-pregnant hamsters, while no effect of pregnancy on the levels of CYP2E RNA was seen. Although rats contain a single CYP2E1 gene transcript. Northern analysis demonstrated the presence of 1.8 and 2.8 kb bands in both liver and lung tissue of the hamster. Pretreatment with ethanol had little effect on the levels of either P450 RNA species in the lungs or livers of nonpregnant, pregnant, and fetal hamsters. These results demonstrate differences in the levels of expression of members of the CYP2E and 2B gene families during pregnancy and late gestation compared to nonpregnant adult hamsters. Fetal animals, like the adults, apparently respond to ethanol treatment by altering the levels of these P450 isozymes at the post-transcriptional level.

Animals↗

PubMed1992

Placental transfer of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone instilled intratracheally in Syrian golden hamsters.

The tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) is a potent tumorigen in adult Syrian golden hamsters and an active transplacental carcinogen in this species. In this study, we have investigated the biodistribution and metabolism of NNK in maternal and fetal hamster tissues as a function of the dose and the time after NNK treatment. Hamsters on day 15 of gestation were instilled intratracheally with single doses (0.05-100 mg/kg) of [5-3H]NNK and sacrificed 30 min later or treated with a single dose (25 mg/kg) of [5-3H]NNK and sacrificed at various times (5-360 min) after treatment. Total radioactivity was quantified in maternal tissues (liver, lung, kidney, placenta, and stomach), in whole fetus and in fetal tissues (liver and lung). NNK and its metabolites were extracted from selected tissues (maternal plasma, amniotic fluid, fetal liver, and lung) and assayed by high-performance liquid chromatography-scintigraphy. Thirty min after treatment, radioactivity associated with NNK and its metabolites showed similar widespread tissue distribution patterns at all doses, with a linear dose relationship observed in whole fetus and fetal tissues. NNK levels detected in maternal plasma, amniotic fluid, fetal liver, and lung were also related linearly to dose. At high doses (25 mg/kg or more) of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol was the major metabolite detected in maternal plasma. Pyridine N-oxidation of NNK predominated at the lowest doses (0.05 and 0.5 mg NNK/kg). The toxicokinetics of NNK demonstrated that this carcinogen is rapidly absorbed from the maternal lung (less than 5 min), metabolized mainly to 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanol, and quickly distributed into the maternal-fetal compartment. Both NNK and its main metabolite 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanol were eliminated slowly from the amniotic fluid, with levels still detectable up to 6 h after NNK treatment. These results demonstrated that NNK instilled intratracheally in pregnant hamsters crossed the placental barrier even at low doses. Moreover, NNK quickly reached fetal tissues and amniotic fluid and was eliminated slowly from these tissues, resulting in an extended exposure of the fetus to this tobacco-specific carcinogen.

Animals↗

PubMed1992

Methods and strategies in lung cancer control.

By the Year 2000, deaths related to lung cancer will increase worldwide to 2 million, mainly due to an increase in cigarette smoking by young adults. The control of lung cancer is a multiphased research and application strategy. The identification of cancer risks and causes and the reduction of carcinogen exposure are the first two phases of this strategy. This paper deals with four interventions with a significant potential impact on the consumption of tobacco. (a) A total ban on the sale of tobacco to minors would be one of the most effective measures to reduce the prevalence of smoking among teenagers and would result, in the long run, in a decrease in lung cancer. (b) In developing countries, the production of tobacco is expected to continue to increase until the Year 2000 and to result in a rapid increase in the incidence of lung cancer. (c) The taxation of tobacco is a powerful tool to reduce smoking, especially among teenagers. (d) In order to reduce the incidence of lung cancer, it is crucial that health professionals inform patients about the health consequences of smoking. Cancer chemoprevention, which could reduce the risk of lung cancer among smokers and exsmokers, is an alternative to primary prevention. The very first step in the chemoprevention strategy is to characterize the chemopreventive agents. The investigator can test its inhibitory capacity in vitro or proceed directly to a tumor inhibition study in laboratory animals. Tumor inhibition studies are carried out at the maximum tolerated dose. Compounds which have demonstrated inhibitory activity are then entered in the next phase of the strategy, the study of chemopreventive efficacies. Chemopreventive agents are best classified according to their mechanism in inhibiting the process of carcinogenesis. One of the most promising approaches to cancer chemoprevention is the use of a combination of chemopreventive agents. The National Cancer Institute funded the development of 14 compounds or combinations of compounds as lung cancer chemopreventive agents. Biomarkers of early lesions are generally used to assess the efficacies of the chemopreventive agents in clinical trials. The biomarker should be able to measure the early phase of the carcinogenic process, be relatively inexpensive, and be the least invasive possible. The biomarkers which have been developed for lung cancer are: index of metaplasia of bronchial tissues and exfoliated atypical bronchial cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Anticarcinogenic Agents↗

PubMed1992

Biodistribution of ellagic acid and dose-related inhibition of lung tumorigenesis in A/J mice.

Ellagic acid (EA), derived from fruit ellagitannins, is known to be antimutagenic and anticarcinogenic in various animal tumor models. In this study, EA at a dose of 4 g/kg diet inhibited multiplicity of tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice by 54%. This inhibition was dose related between 0.06 and 4.0 g/kg diet. In contrast, two related compounds, esculin and esculetin, had no effect on lung tumorigenesis. The biodistribution of EA was studied as a function of dose and time after gavage of EA. The levels of EA in the lung were directly proportional to the dose of EA between 0.2 and 2.0 mmol. The maximum level of EA, corresponding to 21.3 nmol/g, was observed 30 minutes after gavage with 2.0 mmol of EA/kg body wt, which corresponds to only 70 ppm of the administered dose. The levels in liver tissues were 10-fold lower and reached a maximum 30 minutes after gavage. At this interval, the blood level of EA was 1 nmol/ml. The inclusion of EA in cyclodextrin doubles the level of EA in lung tissues. These results demonstrate that EA localizes preferentially in lung tissues and confirm that EA administered orally can inhibit lung tumorigenesis.

Animals↗

PubMed1992

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.

Animals↗

PubMed1992

Effects of sulindac and oltipraz on the tumorigenicity of 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone in A/J mouse lung.

The efficacies of the non-steroidal, anti-inflammatory drug sulindac and the schistosomicidal agent oltipraz in inhibiting lung tumorigenesis was measured in A/J mice. Lung tumors (15.7 tumors/mouse) were induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; 9.1 mg/mouse) administered in drinking water for 7 weeks. Feeding mice with sulindac (123 mg/kg diet), 2 weeks before carcinogen treatment until they were killed reduced tumor multiplicity by 53%. Oltipraz (250 mg/kg diet), however, has no effect on tumorigenesis. The absorption and metabolism of NNK were compared in the stomachs and intestines isolated from mice fed AIN-76A diet or sulindac + diet. Sulindac had no effect on alpha-carbon hydroxylation, pyridine N-oxidation or carbonyl reduction of NNK. Mouse lung explants were cultured with 4.7 microM [5-3H]NNK for 4 or 8 h. The addition of 1 mM sulindac to the culture medium reduces the alpha-carbon hydroxylation and pyridine N-oxidation of NNK. However, the administration of sulindac in the diet prior to the excision of the lung explants had no effect on these two metabolic pathways. We compared the levels of sulindac and its sulfide and sulfone metabolites in the lungs, livers and plasma of mice fed an AIN-76A diet containing 130 mg sulindac/kg for 2 weeks. The sulfide metabolite was the most abundant of the three compounds in plasma (17.6 pmol/microliters) and liver tissues (17.7 pmol/mg) but it could not be detected in lung tissues. These results show that non-steroidal anti-inflammatory drugs constitute a new class of chemopreventive agents in lung tumorigenesis. The tumor chemopreventive activity of sulindac is not mediated by the sulfide metabolite responsible for its anti-inflammatory activity.

Adenoma↗

PubMed1992

Modulation by glutathione of DNA strand breaks induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its aldehyde metabolites in rat hepatocytes.

Activation of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) produced methylating species and two aldehydes: formaldehyde and 4-oxo-4-(3-pyridyl)-butanal (OPB). We investigated the modulation by glutathione of single-strand breaks (SSB) generated by N-methyl-N-nitrosourea (MNU) and the two aldehydes. Hepatocytes were simultaneously exposed to 0.2 mM MNU and to 0-2.00 mM formaldehyde or OPB for 4 h. Both aldehydes induced SSB in a dose-dependent manner. Formaldehyde and OPB exerted a synergistic effect on the formation of DNA SSB by MNU. It is postulated that both aldehydes interfere with DNA repair processes and thus increase the genotoxic effect of DNA methylating species. We investigated whether glutathione (GSH) could protect DNA from NNK-derived intermediates. Formaldehyde (2 mM) and OPB (2 mM) decreased intracellular GSH contents to 60 and 86% of control respectively. DL-Buthionine-[S,R]-sulfoximine (BSO) treatment reduced the GSH contents of hepatocytes to 19% of control but did not reduce the content of cytochrome P450 nor the metabolism of NNK. The frequency of DNA SSB induced by NNK, formaldehyde or OPB was significantly higher in GSH-depleted hepatocytes. GSH repletion with GSH monoethyl ester returned NNK-induced SSB to its initial frequency. OPB but not NNK nor formaldehyde induced double-strand breaks. We conclude that OPB and formaldehyde inhibit the repair of DNA damage induced by methylating species and that GSH reduces the level of DNA damage induced by NNK-derived reactive metabolites.

Analysis of Variance↗

PubMed1991

Metabolism, sister chromatid exchanges, and DNA single-strand breaks induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and their modulation by vitamin A in vitro.

The nicotine-derived N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) is abundant in smokeless tobacco and tobacco smoke and is hepatocarcinogenic in F344 rats. We have investigated how vitamin A modulates sister chromatid exchanges and DNA single-strand breaks induced by NNK. In V79 cells, vitamin A at concentrations ranging from 34.9 to 139.6 microM inhibited sister chromatid exchange frequencies induced by 20 mM NNK activated by primary rat hepatocytes. Sister chromatid exchanges were inhibited by 24, 44, and 55% when cells were cotreated with 34.9, 69.8, and 139.6 microM vitamin A, respectively. DNA single-strand breaks induced by NNK in rat hepatocytes were also inhibited by vitamin A. After 9 h of elution, DNA single-strand breaks induced by 1, 5, and 10 mM NNK were inhibited by 13, 5, and 3.5% in the presence of 69.8 microM vitamin A, respectively. This protective effect by vitamin A was associated with a reduction of alpha-carbon hydroxylation, an activation pathway of NNK. This pathway was inhibited by 50% when cells were cotreated with 3.49 microM vitamin A. The reduction in the hepatic microsomal aminopyrine N-demethylase, aniline hydroxylase, and N,N-dimethyl aniline N-demethylase in the presence of vitamin A (0.035 to 0.35 microM) suggests that vitamin A could reduce NNK genotoxicity by inhibiting the enzymes involved in the activation process.

Animals↗

PubMed1991

Modulation of 4-(methylnitrosamino)-1-(3-pyridyl)-1 butanone demethylation and denitrosation by rat liver microsomes.

The nicotine-derived N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1 butanone (NNK) is abundant in tobacco smoke and is a potent carcinogen in laboratory animals. We have characterized and compared methyl hydroxylation leading to formaldehyde formation (activation pathway) and denitrosation (putative deactivation pathway) of NNK using rat liver microsomes. Methyl hydroxylation was linear between 10 and 50 min and denitrosation was linear between 20 and 65 min. The ratio of nitrite/formaldehyde formation was smaller with 4 mM NNK (0.016) than with N-nitrosodimethylamine (NDMA) (0.037). Liver microsomes exhibited at least two apparent Km values (0.005 and 0.534 mM) for NNK methyl hydroxylation. In vitro methyl hydroxylation of NDMA and NNK were induced by pretreatment of the rat with isopropanol. Butylated hydroxytoluene pretreatment doubled the rate of microsomal denitrosation but did not affect the rate of methyl hydroxylation (+)-Catechin is a flavonoid present in tea, wine and apple skin. We observed that 0.10 mM (+)-catechin inhibited methyl hydroxylation and denitrosation of 4 mM NNK by 40% and 62%, respectively. These results indicated that NNK could be deactivated by denitrosation. Hydroxylation of the NNK methyl group by rat liver microsomes was 34 times more extensive than denitrosation.

Animals↗

PubMed1991

Modulation of the uptake and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by nicotine in hamster lung.

The tobacco-specific carcinogenic nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is formed during the curing and processing of tobacco by nitrosation of nicotine. Nicotine and NNK have structural similarities, and they are both metabolized extensively by lung tissue via several steps known to require oxidative enzyme systems, such as cytochrome P450. On the other hand, nicotine exerts many biological effects similar to those caused by the physiological neurotransmitter acetylcholine, a phenomenon mediated through selective uptake mechanisms via nicotinic cholinergic cell membrane receptors. The aim of this study was to determine if nicotine modulates NKK metabolism in hamster lung explants and if NNK competes with nicotine for binding sites on nicotinic cholinergic receptors in the hamster lung in vivo. Our data show a concentration-dependent inhibition of NNK metabolism in vitro by alpha-carbon hydroxylation and pyridine N-oxidation, whereas the carbonyl reduction of NNK remained unchanged. Radioreceptor assays with membrane receptor fractions of hamster lung after exposure to radiolabeled (S)-(-)-nicotine revealed significant numbers of nicotinic binding sites only in the lungs of hamsters with hyperplasia of pulmonary neuroendocrine cells caused by 4-wk preexposure to hyperoxia. In such animals, radiolabeled nicotine was displaced from the receptor binding sites by NNK. Our data suggest that nicotine can potentially interfere with the carcinogenicity of NNK by competition for enzyme systems essential for the metabolic activation of the nitrosamine and by competition as ligand for nicotinic cholinergic receptors.

Animals↗

PubMed1991

Comparison of DNA alkali-labile sites induced by 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone and 4-oxo-4-(3-pyridyl)butanal in rat hepatocytes.

4-Oxo-4-(3-pyridyl)butanal (OPB) is an aldehyde formed during the activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Using the DNA alkaline elution technique, the properties of DNA alkali-labile sites induced in the isolated rat hepatocytes by NNK and OPB were compared. The DNA single-strand break (SSB) frequencies in vitro, as measured by the elution rate (ER), ranged from 0.015 to 0.479 and were proportional (r2 = 0.991) to the dose (0-2 mM) of OPB. These concentrations, however, were slightly cytotoxic. For example, the LC50 after 4 h of exposure was 2.8 mM. This suggests that OPB-induced DNA SSB result from additive effects of OPB-DNA interaction and the indirect DNA damage associated with OPB cytotoxicity. NNK induced a significant and dose-dependent increase of DNA fragmentation at concentrations ranging from 0.5 to 5.0 mM with ER values ranging from 0.012 to 0.274 (r2 = 0.951). Genotoxicity as measured by the DNA-damaging potency coefficient (DDP) was 810, 345, 131 and 75 for N-methyl-N-nitrosourea (MNU), N-nitrosodimethylamine (NDMA), OPB and NNK respectively. Both MNU- and NNK-induced DNA lesions showed increased lability with increased pH (from 12.1 to 12.5) of the eluting buffer (r2 = 0.979 and 0.967 respectively). In contrast, the number of OPB-induced labile sites were not affected by increases in the pH. These results indicate that OPB is not the metabolite contributing the majority of alkali-labile sites generated by NNK. The filter elution procedure was used to study the in vitro rejoining of SSB in DNA induced by NNK. The extent of DNA SSB rejoining after 18 h of culture of hepatocytes in NNK-free medium were dependent on the concentration of NNK (0.5, 2.0 and 5.0 mM) and ranged from 50 to 90%. Rats were injected s.c. with NNK (0.39 mmol/kg). SSB frequency in liver DNA increased rapidly and reached a maximum 12 h after injection. DNA SSB frequency declined during the next 2 weeks with biphasic kinetics. The fast phase (75% rejoining of DNA SSB between 12 h and 2 days) was followed by a slow one (25% of DNA SSB maintained during the next 5 days but not present after 2 weeks). The results of this study better define the role of OPB-induced DNA damage. The persistence of DNA SSB in the liver of NNK-treated rats reflects the inability of this tissue to repair all DNA lesions.

Analysis of Variance↗

PubMed1991

Protective effects of vitamin A against the genotoxicity of NNK, a nicotine-derived N-nitrosamine.

In this investigation, the effect of vitamin A on 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced genotoxic damage in rat liver was studied and correlated with NNK metabolism and DNA single strand breaks (DNA-SSBs). Male adult Fischer rats were fed a vitamin A deficient diet for 8 weeks and then divided into two sub-groups. Rats in group Ia and group Ib were fed vitamin A supplemented and vitamin A deficient diets respectively for an additional 4 weeks. Rats were then treated with a single i.p. injection of NNK (25-100 mg/kg) and subjected to partial hepatectomy 1 week later. After 72 h, liver perfusions were performed for cell preparation. Non-hepatectomized rats were used as controls (group IIa and IIb, consisting of rats fed vitamin A supplemented and deficient diets respectively). In non-hepatectomized rats (groups IIa and IIb), micronucleus (MN) frequencies were not significantly increased. This confirms the importance of cell proliferation for MN formation. In rats fed a vitamin A deficient diet and subjected to partial hepatectomy (group Ib), a significant increase of MN was observed (2.2 +/- 0.3, 5.7 +/- 0.9, 12.8 +/- 3.8 and 21.2 +/- 4.3% for control, 25, 50 and 100 mg NNK/kg body wt respectively). In rats fed a vitamin A supplemented diet (group Ia) we have observed a significant decrease of MN induction, as compared to rats fed a vitamin A deficient diet (group Ib) (2.7 +/- 1.0, 2.9 +/- 0.9, 9.7 +/- 2.7, 14.8 +/- 3.2% for control, 25, 50 and 100 mg NNK/kg body wt respectively). Freshly isolated hepatocytes from rats fed vitamin A deficient and supplemented diets but without NNK treatment in vivo were used to study NNK metabolism and DNA-SSBs. Results showed that hepatocytes isolated from rats fed a vitamin A supplemented diet (group C) metabolized NNK less effectively than hepatocytes isolated from rats fed a vitamin A deficient diet (group B) (NNK metabolism was decreased by 0.5-fold in group C as compared to group B). This inhibition was observed only with the NNK activation pathway, alpha-carbon hydroxylation, but not with a deactivation pathway: pyridine-N-oxidation. DNA-SSBs induced by NNK were also reduced in hepatocytes isolated from rats fed a vitamin A supplemented diet as compared to hepatocytes isolated from rats fed a deficient diet. These reductions were 7.8, 12.4 and 4.5% after 6 h of elution and 9.1, 8.5 and 3.0% after 9 h of elution for 1, 5 and 10 mM NNK respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Inhibition of the metabolism and genotoxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat hepatocytes by (+)-catechin.

(+)-Catechin is a plant flavonoid which decreases the mutagenicity of several mutagens and carcinogens. In this study, we have investigated how (+)-catechin could inhibit the metabolism and DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen. Addition of 5 to 1000 microM (+)-catechin to rat hepatocytes cultured with 4.5 mM NNK caused a concentration-dependent reduction of alpha-carbon hydroxylation which is the activation pathway of NNK. Under the same conditions, (+)-catechin had a less significant effect on pyridine N-oxidation, which is a deactivation pathway. Reduction of the carbonyl group of NNK was not inhibited by (+)-catechin. We had previously shown that NNK induced single-strand breaks (SSBs) in primary culture of hepatocytes. In this study, we observed that 1.0 mM (+)-catechin inhibited the DNA SSBs induced by 1 mM NNK by 31%. With 1 mM N-nitrosodimethylamine, the inhibition of DNA SSBs was 30%. We concluded that (+)-catechin selectively inhibits the enzymes involved in the activation of NNK. Rats were gavaged with (+)-catechin (1.5 mmol/kg), injected s.c. 1 h later with NNK (0.39 mmol/kg) and killed 4 h after NNK treatment. (+)-Catechin significantly reduced DNA SSBs induced by NNK. Rats were injected s.c. with 0.39 mmol/kg NNK. (+)-Catechin reduced the methylation of liver DNA at the O6-guanine and N-7 guanine sites by 28 and 34% respectively. These results demonstrate that (+)-catechin inhibits the formation of DNA-damaging intermediates by selectively impairing the enzymatic activation of NNK. They suggest that (+)-catechin could be an effective preventive agent against NNK hepatocarcinogenicity.

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