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C J Michejda

Publications and source records attributed to C J Michejda.

At least 55 records · Page 3Linked to original sources
PubMed1985

Metabolic studies of 15N-labeled N-nitrosoproline in isolated rat hepatocytes and subcellular fractions.

The metabolism of the non-carcinogenic N-nitrosoproline (NPRO) was investigated in vitro using both S9 preparations and isolated hepatocytes from F344 rats. The studies were performed using 15N-labeled nitrosamine and the reaction mixtures were examined mass spectrometrically for the presence of 15N2 or other 15N-labeled gaseous products. In addition, the metabolism of NPRO was monitored by capillary gas chromatography. The results indicated no 15N2 production from either the hepatocyte or S9 preparations, as well as no detectable loss of substrate from the reaction mixtures. Mass spectrometric analysis failed to reveal any metabolites of NPRO. The results suggest that NPRO may be refractory to the normal nitrosamine activating enzymes, confirming its suitability for use in human epidemiological studies of endogenous nitrosation.

Animals↗
PubMed1985

Use of 3,4-dichlorobenzenethiol as a trapping agent for alkylating intermediates during in vitro metabolism of nitrosamines.

Our studies using 3,4-dichlorobenzenethiol as a probe for methylating agent production during exposure of N-nitrosodimethylamine to rat liver S-9 preparations produced results different from those of an investigation reported in the literature. Methyl-3,4-dichlorophenyl thioether was detected, but the quantities found were not significantly different from the background levels of methylation product detected in the absence of nitrosamine. Only about 10% of the thioether isolated after incubating N-nitrosodi[14C]methylamine as substrate was radioactive. The results indicate that the majority of the methyl groups transferred to the sulfur nucleophile in our experiments came from components of the incubation mixture other than the nitrosamine. Some artifactual methylation was also associated with the analytical procedure. We conclude that 3,4-dichlorobenzenethiol should be used with caution in studies of alkylation during the in vitro metabolism of carcinogenic nitrosamines.

Animals↗
PubMed1984

Metabolism of 15N-labelled N-nitrosodimethylamine and N-nitroso-N-methylaniline by isolated rat hepatocytes.

The N-demethylation of 15N-labeled N-nitrosodimethylamine (DMN) and N-nitroso-N-methylaniline (NMA) by isolated rat hepatic cells has been investigated. The values obtained in this system for molecular nitrogen formed during metabolism, compared with substrate consumed, were DMN 47%, NMA 23%, and N-nitroso-N-methylurea (NMU) 105%. The results for DMN are roughly halfway between those previously determined with rat liver S-9 fraction in vitro (33%) and in vivo (67%). For NMA, the hepatocyte data are closer to those obtained from S-9 in vitro (19%), rather than the in vivo (52%). No mixed nitrogen ( 15N14N ) or labeled nitrogen oxides were found.

Animals↗
PubMed1984

Photochemistry of N-nitrosamines in neutral media.

N-Nitrosamines have long been held to be photostable in solution in absence of acid, although a few reports to the contrary have been published. This paper reports a study of the photolysis of N-nitrosopiperidine and N-nitrosodiisopropylamine in aprotic and protic solvents. It is shown that the cleavage of the N-N bond is a very facile process. In the absence of radical scavengers, however, reformation of the nitrosamines is very rapid, giving little or no net photolysis. In the presence of scavengers, photolysis proceeds with high efficiency to give non-nitrosamine products. When the trapping agent is oxygen, the net reaction is photo-oxygenation to the corresponding nitramine. The nitramine, in turn, is photolabile and undergoes photolysis to give cleavage products. The formation of the nitramine proceeds through the initial photochemical cleavage of the nitrosamine to the aminyl radical and nitric oxide, followed by the oxidation of the nitric oxide to nitrogen dioxide and the recombination of the latter with the aminyl radical. These results have some important implications for the destruction of nitrosamine wastes.

Hydrogen-Ion Concentration↗
PubMed1984

alpha-Hydroxylation of nitrogen-15 labelled N-nitrosamines by isolated hepatocytes.

The principal pathway of nitrosamine metabolism has long been considered to be alpha-hydroxylation. For N-nitrosodialkylamines, this hypothesis requires that a molecule of molecular nitrogen be released for every molecule of nitrosamine that is alpha-hydroxylated. Thus, the quantitative determination of nitrogen formation should provide a measure of the importance of this pathway. This method was applied earlier to the doubly-labelled nitrogen-15 compounds, N-nitrosodimethylamine (NDMA), N-nitrosomethylphenylamine (NMPhA) and N-methyl-N-nitrosourea (MNU), using both a 9 000 X g supernatant fraction of liver and the intact animal as metabolic systems. The in-vitro results were quite different from those obtained in vivo. The majority of the NDMA (67%) and the MNU (88%) were converted to nitrogen in vivo, while NMPhA gave considerably less nitrogen (52%). These results differed by a factor of approximately two from those obtained in vitro (NDMA, 33%; NMPhA, 18.8% and MNU, 96%). Since such differences may be a result of the loss of cellular architecture, we have extended the work to include isolated hepatocytes. It had been shown previously that isolated hepatocytes constitute a practical alternative to in-vivo systems, even though the correlation with in-vivo metabolism appears to depend on the substrate analysed. The values obtained using this system (NDMA, 47%; NMPhA, 23%; and MNU 105%) reconfirm that metabolism may be substrate dependent. As in our previous studies, no mixed nitrogen (15N14N) or labelled nitrogen oxides were found. The data are all consistent with the hypothesis that at least one demethylase for each of the nitrosamine substrates is associated with a cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

The effect of substituents in the aromatic ring on carcinogenicity of N-nitrosomethylaniline in F344 rats.

N-Nitroso-N-methylaniline (NMA) and N-nitroso-N-methyl-4-fluoroaniline (p-F-NMA), both non-mutagenic in Salmonella typhimurium and N-nitroso-N-methyl-4-nitroaniline (p-NO2-NMA), a potent mutagen, were tested for carcinogenicity in F344 rats. NMA was shown to induce a high level of tumors in the upper gastrointestinal tract, particularly in the esophagus. Male rats treated with NMA died with tumors at a slightly higher rate than females, although the final tumor yield was the same. Most of the rats treated with p-F-NMA also developed tumors of the esophagus, but they died less rapidly than the NMA treated rats, indicating that p-F-NMA is a slightly weaker carcinogen than NMA. The powerful, directly acting mutagen, p-NO2-NMA did not appear to induce tumors at all since its tumor spectrum was essentially identical to that of the untreated control rats. Thus, the carcinogenic activities of NMA and its substituted analogs do not appear to correlate with bacterial mutagenesis assays. Additionally, NMA, p-F-NMA and N-nitroso-N-methyl-4-bromoaniline, the last a strong mutagen in S. typhimurium, were shown not to induce sister chromatid exchanges in CHO cells and in a clone of a CHO:liver cell hybrid which had previously been shown to be sensitive to chemical agents which require metabolic activation.

Aflatoxin B1↗
PubMed1982

O-alkylation of N-nitrosamines. Transalkylation of N-nitrosamines and formation of electrophilic intermediates.

Ring closure of appropriately beta-substituted nitrosamines leads to the formation of dihydrooxadiazolium ions. These relatively stable, crystalline compounds react with nucleophiles, either at the position of the beta-substituent or by displacement of an alkyl group bound to the nitrogen. These compounds are intramolecular varients of alkoxydiazenium ions, which are formed by the O-alkylation of nitrosamines with a variety of agents, including alkyl-fluorosulfonates. The alkoxydiazenium ions are also stable, crystalline solids. They react with nucleophiles usually by displacement at the O-alkyl group. One exception to this, however, is the reaction of 3,4-dichlorothiopheno in organic solvents to form thioethers by O-dealkylation and N-dealkylation. The by-products of the dealkylations are the corresponding nitrosamines. The aqueous reactions of 3,4-dichlorothiophenol are slower and pH-dependent. In acidic media, 3,4-dichlorothiophenol reacts only at the O-alkyl groups, whereas in weakly alkaline media, a little O- and N-dealkylation is observed, together with considerable base-catalysed decomposition of the alkoxydiazenium ions.

Alkylation↗
PubMed1981

Mutagenicity and rat-liver S9 demethylation kinetics of N-nitrosomethylaniline and its ring-substituted derivatives.

N-nitrosomethylaniline, an esophageal carcinogen in the rat, was inactive in the Ames mutagenicity assay under all conditions examined. However, various ring-substituted N-nitrosomethylanilines were found to be mutagenic; some of these did not require activation. No correlation could be found between the rate of demethylation or substituent effects of the ring-substituted N-nitrosomethylanilines and mutagenic potency. The protein concentration and levels of aryl hydrocarbon hydroxylase in the liver S9 fraction were determined to be poor indices of the ability of S9 to activate nitrosamines to their mutagenic metabolites.

Animals↗
PubMed1981

alpha-Hydroxylation pathway in the in vitro metabolism of carcinogenic nitrosamines: N-nitrosodimethylamine and N-nitroso-N-methylaniline.

Evolution of 15N2-labeled molecular nitrogen was used to gauge the extent of alpha-hydroxylation during rat liver homogenate metabolism of doubly 15N-labeled N-nitrosodimethylamine (DMN) and N-nitrosomethylaniline (NMA). These measurements were correlated with the extent of total metabolism as measured by the disappearance of the nitrosamines and by the formation of formaldehyde. The results indicate that approximately 34% of DMN and 19% of NMA were metabolized by the alpha-hydroxylation pathway. Positive controls utilizing doubly 15N-labeled N-nitroso-N-methylurea yielded 96% of labeled nitrogen. These results are in variance with previously published data which claimed that either less than 5% or about 100% of DMN is metabolized by that route in vitro. Formaldehyde formation was shown to be a poor measure of the extent of metabolism. Semicarbazide gave rise to both formaldehyde and nitrogen, which makes it an undesirable component of the in vitro metabolism mixtures, particularly when those two substances are being measured.

Animals↗
PubMed1980

Mutagenicity of trialkyltriazenes: mutagenic potency of alkyldiazonium ions, the putative ultimate carcinogens from dialkylnitrosamines.

Although aryldialkyltriazenes have been known for many years and their mutagenic, carcinogenic and carcinostatic properties have been investigated, almost nothing is known of the related trialkyltriazenes. Our recently developed general preparative route to these substances has allowed the examination of the mutagenic properties of several representative examples of this class of compounds. This, 1-benzyl-3,3-dimethyl-, 3-benzyl-1,3-dimethyl, 3-benzyl-3-methyl-1-n-butyl- and 1,3-di-n-butyl-3-methyltriazenes are direct acting mutagens in the TA1535 strain of Salmonella typhimurium. The respective mutagenic potencies of these substances can be accounted for by the in situ generation of alkyldiazonium ions. These ions are considered to be strong candidates for the ultimate mutagens/carcinogens derived from some dialkylnitrosamines.

Mutagenicity Tests↗
PubMed1980

Carcinogenicity of the isomeric, N-nitroso-delta3-and N-nitroso-delta2-piperidines in rats and the in vivo isomerization of the delta3-to the delta2-isomer.

N-Nitroso-1,2,3,6-tetrahydropyridine (N-nitroso-delta3-piperidine), N-nitroso-1,2,3,4-tetrahydropyridine (N-nitroso-delta2-piperidine) and N-nitroso-3,4-epoxypiperidine were tested for carcinogenicity in Fischer 344 rats. The unsaturated nitrosamines were administered in drinking water (100 mg/l). The epoxide was administered by gavage in corn oil (11.5 mg/ml, 0.2 ml twice a week). Both of the unsaturated nitrosamines were potent carcinogens (most of the animals died by the 35th week), and both produced many esophageal tumors, a property which they have in common with the parent compound, N-nitrosopiperidine. The spectrum of the other tumors formed, however, was different. The delta3-isomer produced hemangioendothelial sarcomas in the liver, which were absent in the tumor spectrum of the delta2-isomer and N-nitrosopiperidine. The delta2-isomer, on the other hand, produced tumors of the forestomach and the oropharynx, which were essentially absent in the rats treated with the delta3-isomer. N-nitroso-3,4-epoxypiperidine was a toxic compound (8 deaths in the first 5 weeks), but most of the remaining animals survived to 40 weeks. Of these, 8 animals died of induced tumors (esophagus and liver). The delta2- and the delta3-isomers were administered by gavage to groups of rats and the blood of these animals was withdrawn at timed intervals. Analysis of the serum revealed that both of the nitrosamines were cleared rapidly from circulation but that at the same time the delta3-isomer was being isomerized to the delta2. The reverse transformation did not occur in vivo.

Animals↗
PubMed1980

Formation and chemistry of alpha- and beta-oxidized nitrosamines.

Nitrosamines, in general, require metabolic transformation to produce electrophilic metabolites. These interact with cellular nucleophiles to initiate the chain of events culminating in tumour initiation. The enzymatic hydroxylation of the alpha-carbon of nitrosamines has long been held to be an important process in the formation of carcinogenic metabolites. The present work suggests, however, that there may be other processes which also result in the formation of electrophilic species. These include: (1) beta-hydroxylation, followed by appropriate conjugation, which results in the formation of electrophilic oxadiazolium ions. (2) O-alkylation of nitrosamines, which has the effect of labilizing the alkyl groups attached to the nitrogen toward nucleophilic substitution. (3) The formation of alpha, beta-unsaturated nitrosamine (N-nitrosoenamines), which are highly reactive species that can be readily transformed to alpha-hydroxylated nitrosamines by acid-catalyzed hydration. N-nitrosoenamines also react directly with a variety of nucleophilic reagents. The isomerization of beta, gamma-unsaturated nitrosamines to the alpha, beta-unsaturated isomers has been demonstrated to occur in vivo.

Alkylation↗
PubMed1979

Derivatives of side-chain hydroxylated nitrosamines direct acting mutagens in Salmonella typhimurium.

Methyl-(beta-tosyloxyethyl)nitrosamine and 3-methyl-4,5-dihydro-1,2,3-oxadiazolium tosylate are potent direct acting mutagens in the Ames assay, as is N-nitrosoprolinyl tosylate. These compounds are derived from beta-hydroxylated nitrosamines. The closely related methyl-(gamma-tosyloxypropyl)nitrosamine is not mutagenic without activation. These data are consistent with the chemical behavior of these substances, which suggest that suitable derivatives of beta-hydroxylated nitrosamines, such as O-sulfates, may be direct-acting biological alkylating agents.

Dose-Response Relationship, Drug↗
PubMed1979

Evidence for several demethylase enzymes in the oxidation of dimethylnitrosamine and phenylmethylnitrosamine by rat liver fractions.

The steady state kinetics and isotope effects were examined for demethylation of dimethylnitrosamine and phenylmethylnitrosamine, as well as their deuterated analogs, using the S-9 fraction, the microsomal pellet, and postmicrosomal supernatant from rat livers. The isotope effect (ratio of maximal rates for the deuterated and light substrates) using the S-9 from Long-Evans rat livers was found to be 1.82 for dimethylnitrosamine and 5.38 for phenylmethylnitrosamine. Phenobarbital was shown to induce dimethylnitrosamine demethylase activity in the microsomal pellet of both Long-Evans and Sprague-Dawley rats but to repress this activity in the postmicrosomal supernatant in the Long-Evans rats, while markedly increasing it in the Sprague-Dawley rats. It was also found that there was nitrosamine demethylase activity in the so-called "pH 5 enzymes" and in the supernatant from that preparation. The latter activity shows substantially different characteristics from that found in the other fractions.

Animals↗
PubMedDate not supplied

Evidence for an unstable DNA adduct from N-nitroso-N-methylaniline.

N-Nitroso-N-methylaniline (NMA) is an esophageal carcinogen in F344 rats. Attempts to detect binding of NMA to DNA or RNA have not been successful. NMA is not mutagenic in the standard Ames bacterial assay, and it did not induce sister chromatid exchanges in mammalian cells. NMA forms the benzenediazonium ion (BDI) during metabolism. This ion has been known to react with aromatic amines, such as adenine, to form triazene coupling products. The purpose of this research was to demonstrate that a triazene adduct, which would be expected to be hydrolytically unstable, was formed by coupling with the adenine residues in DNA. Liver DNA from a rat treated with NMA or from in vitro reactions of BDI with DNA was treated with sodium borohydride. This reaction was shown to result in the reduction of 6-(1-phenyltriazeno)purine to 6-hydrazinopurine (N6-aminoadenine). The hydrolysate of the DNA, presumably containing the hydrazine, was treated with 4-(dimethylamino)naphthaldehyde, and the resulting hydrazone was isolated by reverse-phase HPLC using fluorescence detection. The identity of the adduct was demonstrated by high-resolution mass spectrometry. These data suggest strongly that NMA forms an unstable triazene adduct with adenine in DNA both in vitro and in vivo.

Animals↗
PubMedDate not supplied

Stereoselectivity in the microsomal conversion of N-nitrosodimethylamine to formaldehyde.

The possibility that N-nitrosodimethylamine (NDMA) might be metabolized preferentially at either the syn (relative to the nitroso oxygen) or the anti methyl group has been examined by comparing the rates of formaldehyde production when unlabeled NDMA, its fully deuteriated analogue (NDMA-d6), and (Z)- or (E)-N-nitrosomethyl(methyl-d3)amine (NDMA-d3) were incubated in turn at concentrations of 0-2.4 mM with acetone-induced rat liver microsomes. The Km values for the conversion of (Z)- and (E)-NDMA-d3 to formaldehyde were identical to each other within experimental error (32 +/- 2 and 35 +/- 1 microM, respectively) but different from those for NDMA (24 +/- 6 microM) and NDMA-d6 (116 +/- 3 microM); similar Vmax values were observed for the four isotopic variants [7.5-8.1 nmol/(mg of protein.min)]. The observed similarity of kinetic parameters for (Z)- and (E)-NDMA-d3 suggested that the isotopic composition of the methyl group is an energetically more important determinant of its rate of oxidation at the NDMA demethylase active site than is its orientation relative to the nitroso oxygen atom. The absence of syn vs anti stereospecificity was confirmed via product isolation studies, in which the formaldehyde generated from each of the four isotopomers was trapped as the dimedone adduct and assayed for deuterium content by mass spectrometry; again, a strong preference for metabolism at CH3 vs CD3 regardless of stereochemistry was observed, though the data on CH2O generation suggested that there may be a slight net excess of anti attack. The results indicate that the microsomal enzymes employed display little regioselectivity in metabolizing the syn vs anti methyl groups of NDMA.

Animals↗
PubMedDate not supplied

1,3-Dialkyltriazenes: reactive intermediates and DNA alkylation.

The reactions of calf thymus (ct) DNA with 1,3-dimethyltriazene (DMT), N-methyl-N-nitrosourea (MNU), 1,3-diethyltriazene (DET), N-ethyl-N-nitrosourea (ENU), and 1-ethyl-3-methyltriazene (MET) were studied as a function of concentration of the alkylating agents, of various buffers, and of ionic strength. The amount of alkylation at the 7- and O6-positions of guanine increased linearly with dose over a 10-fold concentration range. The slopes of the DMT and MNU curves were identical as were those of DET and ENU. These data suggest that both types of compounds alkylate DNA via a similar intermediate, presumably the corresponding alkanediazonium ion. MET methylates and ethylates DNA, the amount of each product being a function of the competitive formation of the two diazonium ions possible from MET. The MET product ratios could be reproduced by an appropriate mixture of DET and DMT. The alkylation of DNA by DMT and by MET is very sensitive to ionic strength, to the nature of the buffer, and to the identity of the salt used to balance ionic strength. In general, the reaction is favored by low ionic strength, by amine rather than oxy acid buffers, and by doubly charged inert anions. The alkylation of DNA is inversely proportional to the logarithm of the ionic strength over a wide range. The mutagenic activity of triazenes in Salmonella typhimurium is correlated very well with the ability of the triazenes to form adducts, particularly O6-guanine adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗