N-nitrosamines

bacterial mutagenesis and in vitro metabolism

Research output: Contribution to journalArticle

Abstract

Many nitrosamines are potent mutagens. The rate-limiting step in their in vitro metabolism to mutagens is usually a single enzymatic reaction catalyzed by one or more of the many cytochrome P-450-dependent mixed-function oxidases present in the microsomal cell fraction. Current evidence indicates that this reaction activates nitrosamines to α-hydroxynitrosamines, which have half-lives on the order of seconds. This product decomposes to an aldehyde and a much shorter-lived ultimate metabolite which is probably an alkyl diazonium ion or an alkyl carbocation. This may react with DNA leading to premutagenic adducts. Such adducts represent a very small fraction of the ultimate mutagen, with the rest reacting with water to yield the corresponding alcohol. Evidence for this pathway includes (1) the observation of deuterium isotope effects in metabolism and mutagenesis, (2) products (aldehydes, alcohols, and N2) consistent with this pathway, (3) studies on metabolism of nitrosamines using purified cytochrome P-450, (4) formation of DNA adducts such as O6-alkylguanines which are consistent with those expected from the ultimate mutagen, (5) expected products and genotoxic effects of other sources of activated nitrosamines, e.g., α-acetoxynitrosamines, alkanediazotates and related compounds. Hydroxylation of nitrosamines at other positions also occurs in vitro (usually to a lesser extent), but these products are generally stable and must be further metabolized to exert mutagenic effects (with the exception of N-nitrosoalkyl(formylmethyl)amines, which are direct-acting mutagens). Because only low percentages of nitrosamines are metabolized in vitro, the contribution to mutagenesis by secondary metabolism is small. In this respect, in vitro metabolism can differ significantly from in vivo metabolism. Bacterial mutagenesis by nitrosamines has most often been studied in Salmonella typhimurium and to a lesser extent E. coli. Mutagenesis by nitrosamines generally requires a source of microsomes (a 9000 × g supernatant fraction is often used), and NADPH. Liver fractions from Aroclor-1254- or PB-induced rodents have been most frequently employed but liver fractions from untreated animals, and homogenates of other organs (lung, kidney, nasal mucosa, and pancreas) have also been utilized. Liver homogenates from humans are generally similar to those from untreated rats in metabolizing nitrosamines to mutagens but large interindividual variations are observed. Mutagenesis is often most effective using a liquid preincubation, a slightly acidic incubation mixture and hamster liver fractions. Base-pair substitution mutants are generally most sensitive to the mutagenic effects of many nitrosamines, and mutagenesis is often enhanced in Uvr- strains and by error-prone DNA repair processes. The higher homologue di-n-alkylnitrosamines tend to induce mutagenesis at lower doses than the lower homologues, but revertant numbers level off and decline at much lower values than those from the lower homologues. Higher homologue N-nitrosomethylalkylamines are particularly potent over a large dose range probably because they are rapidly metabolized to methyl diazonium ion, and higher homologue cyclic nitrosamines are quite potent and tend to be more potent than lower homologues. Mutagenicity by most nitrosamines drops off dramatically when the number of carbons in the compounds exceeds 12-14. Hydroxyl and keto groups generally reduce mutagenic activity but deactivation is strongly dependent on position. At least part of the reduction in mutagenesis is due to a reduced rate of metabolism. Deuterium substitution and branching at the α-carbons reduce mutagenic activity and incorporation of most polar groups and fluorines into nitrosamines greatly reduces mutagenic activity. Phenylating nitrosamines are very weak mutagens at best in most of the common tester strains, but are moderately potent in TA104, which can detect mutations at AT base pairs. Substitution of β-chlorines and 2,3-double bonds usually increases mutagenic activity. Nitrosamines where the α-positions are blocked, e.g., α,α,α',α'-tetramethylnitrosamines are nonmutagenic, as are nitrosamines which give rise to tert.-butyl diazonium ion. Most carcinogenic nitrosamines have been reported to be mutagenic in one or more laboratories (with considerable interlaboratory variation), and a low percentage of noncarcinogenic nitrosamines are mutagenic. Correlations between mutagenic and carcinogenic potencies were only observed in several studies.

Original languageEnglish (US)
Pages (from-to)81-134
Number of pages54
JournalMutation Research/Reviews in Genetic Toxicology
Volume186
Issue number2
DOIs
StatePublished - 1987

Fingerprint

Nitrosamines
Mutagenesis
Metabolism
Mutagens
Liver
In Vitro Techniques
Substitution reactions
Deuterium
Ions
Aldehydes
Base Pairing
Cytochrome P-450 Enzyme System
Carbon
Alcohols
Chlorodiphenyl (54% Chlorine)
Secondary Metabolism
Hydroxylation
Salmonella
Fluorine
DNA Adducts

Keywords

  • Cytochrome P-450-dependent
  • Mixed-function oxidases
  • N-Nitrosamines
  • Rate-limiting step in metabolism in vitro

ASJC Scopus subject areas

  • Genetics
  • Toxicology
  • Medicine(all)

Cite this

N-nitrosamines : bacterial mutagenesis and in vitro metabolism. / Guttenplan, Joseph.

In: Mutation Research/Reviews in Genetic Toxicology, Vol. 186, No. 2, 1987, p. 81-134.

Research output: Contribution to journalArticle

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N2 - Many nitrosamines are potent mutagens. The rate-limiting step in their in vitro metabolism to mutagens is usually a single enzymatic reaction catalyzed by one or more of the many cytochrome P-450-dependent mixed-function oxidases present in the microsomal cell fraction. Current evidence indicates that this reaction activates nitrosamines to α-hydroxynitrosamines, which have half-lives on the order of seconds. This product decomposes to an aldehyde and a much shorter-lived ultimate metabolite which is probably an alkyl diazonium ion or an alkyl carbocation. This may react with DNA leading to premutagenic adducts. Such adducts represent a very small fraction of the ultimate mutagen, with the rest reacting with water to yield the corresponding alcohol. Evidence for this pathway includes (1) the observation of deuterium isotope effects in metabolism and mutagenesis, (2) products (aldehydes, alcohols, and N2) consistent with this pathway, (3) studies on metabolism of nitrosamines using purified cytochrome P-450, (4) formation of DNA adducts such as O6-alkylguanines which are consistent with those expected from the ultimate mutagen, (5) expected products and genotoxic effects of other sources of activated nitrosamines, e.g., α-acetoxynitrosamines, alkanediazotates and related compounds. Hydroxylation of nitrosamines at other positions also occurs in vitro (usually to a lesser extent), but these products are generally stable and must be further metabolized to exert mutagenic effects (with the exception of N-nitrosoalkyl(formylmethyl)amines, which are direct-acting mutagens). Because only low percentages of nitrosamines are metabolized in vitro, the contribution to mutagenesis by secondary metabolism is small. In this respect, in vitro metabolism can differ significantly from in vivo metabolism. Bacterial mutagenesis by nitrosamines has most often been studied in Salmonella typhimurium and to a lesser extent E. coli. Mutagenesis by nitrosamines generally requires a source of microsomes (a 9000 × g supernatant fraction is often used), and NADPH. Liver fractions from Aroclor-1254- or PB-induced rodents have been most frequently employed but liver fractions from untreated animals, and homogenates of other organs (lung, kidney, nasal mucosa, and pancreas) have also been utilized. Liver homogenates from humans are generally similar to those from untreated rats in metabolizing nitrosamines to mutagens but large interindividual variations are observed. Mutagenesis is often most effective using a liquid preincubation, a slightly acidic incubation mixture and hamster liver fractions. Base-pair substitution mutants are generally most sensitive to the mutagenic effects of many nitrosamines, and mutagenesis is often enhanced in Uvr- strains and by error-prone DNA repair processes. The higher homologue di-n-alkylnitrosamines tend to induce mutagenesis at lower doses than the lower homologues, but revertant numbers level off and decline at much lower values than those from the lower homologues. Higher homologue N-nitrosomethylalkylamines are particularly potent over a large dose range probably because they are rapidly metabolized to methyl diazonium ion, and higher homologue cyclic nitrosamines are quite potent and tend to be more potent than lower homologues. Mutagenicity by most nitrosamines drops off dramatically when the number of carbons in the compounds exceeds 12-14. Hydroxyl and keto groups generally reduce mutagenic activity but deactivation is strongly dependent on position. At least part of the reduction in mutagenesis is due to a reduced rate of metabolism. Deuterium substitution and branching at the α-carbons reduce mutagenic activity and incorporation of most polar groups and fluorines into nitrosamines greatly reduces mutagenic activity. Phenylating nitrosamines are very weak mutagens at best in most of the common tester strains, but are moderately potent in TA104, which can detect mutations at AT base pairs. Substitution of β-chlorines and 2,3-double bonds usually increases mutagenic activity. Nitrosamines where the α-positions are blocked, e.g., α,α,α',α'-tetramethylnitrosamines are nonmutagenic, as are nitrosamines which give rise to tert.-butyl diazonium ion. Most carcinogenic nitrosamines have been reported to be mutagenic in one or more laboratories (with considerable interlaboratory variation), and a low percentage of noncarcinogenic nitrosamines are mutagenic. Correlations between mutagenic and carcinogenic potencies were only observed in several studies.

AB - Many nitrosamines are potent mutagens. The rate-limiting step in their in vitro metabolism to mutagens is usually a single enzymatic reaction catalyzed by one or more of the many cytochrome P-450-dependent mixed-function oxidases present in the microsomal cell fraction. Current evidence indicates that this reaction activates nitrosamines to α-hydroxynitrosamines, which have half-lives on the order of seconds. This product decomposes to an aldehyde and a much shorter-lived ultimate metabolite which is probably an alkyl diazonium ion or an alkyl carbocation. This may react with DNA leading to premutagenic adducts. Such adducts represent a very small fraction of the ultimate mutagen, with the rest reacting with water to yield the corresponding alcohol. Evidence for this pathway includes (1) the observation of deuterium isotope effects in metabolism and mutagenesis, (2) products (aldehydes, alcohols, and N2) consistent with this pathway, (3) studies on metabolism of nitrosamines using purified cytochrome P-450, (4) formation of DNA adducts such as O6-alkylguanines which are consistent with those expected from the ultimate mutagen, (5) expected products and genotoxic effects of other sources of activated nitrosamines, e.g., α-acetoxynitrosamines, alkanediazotates and related compounds. Hydroxylation of nitrosamines at other positions also occurs in vitro (usually to a lesser extent), but these products are generally stable and must be further metabolized to exert mutagenic effects (with the exception of N-nitrosoalkyl(formylmethyl)amines, which are direct-acting mutagens). Because only low percentages of nitrosamines are metabolized in vitro, the contribution to mutagenesis by secondary metabolism is small. In this respect, in vitro metabolism can differ significantly from in vivo metabolism. Bacterial mutagenesis by nitrosamines has most often been studied in Salmonella typhimurium and to a lesser extent E. coli. Mutagenesis by nitrosamines generally requires a source of microsomes (a 9000 × g supernatant fraction is often used), and NADPH. Liver fractions from Aroclor-1254- or PB-induced rodents have been most frequently employed but liver fractions from untreated animals, and homogenates of other organs (lung, kidney, nasal mucosa, and pancreas) have also been utilized. Liver homogenates from humans are generally similar to those from untreated rats in metabolizing nitrosamines to mutagens but large interindividual variations are observed. Mutagenesis is often most effective using a liquid preincubation, a slightly acidic incubation mixture and hamster liver fractions. Base-pair substitution mutants are generally most sensitive to the mutagenic effects of many nitrosamines, and mutagenesis is often enhanced in Uvr- strains and by error-prone DNA repair processes. The higher homologue di-n-alkylnitrosamines tend to induce mutagenesis at lower doses than the lower homologues, but revertant numbers level off and decline at much lower values than those from the lower homologues. Higher homologue N-nitrosomethylalkylamines are particularly potent over a large dose range probably because they are rapidly metabolized to methyl diazonium ion, and higher homologue cyclic nitrosamines are quite potent and tend to be more potent than lower homologues. Mutagenicity by most nitrosamines drops off dramatically when the number of carbons in the compounds exceeds 12-14. Hydroxyl and keto groups generally reduce mutagenic activity but deactivation is strongly dependent on position. At least part of the reduction in mutagenesis is due to a reduced rate of metabolism. Deuterium substitution and branching at the α-carbons reduce mutagenic activity and incorporation of most polar groups and fluorines into nitrosamines greatly reduces mutagenic activity. Phenylating nitrosamines are very weak mutagens at best in most of the common tester strains, but are moderately potent in TA104, which can detect mutations at AT base pairs. Substitution of β-chlorines and 2,3-double bonds usually increases mutagenic activity. Nitrosamines where the α-positions are blocked, e.g., α,α,α',α'-tetramethylnitrosamines are nonmutagenic, as are nitrosamines which give rise to tert.-butyl diazonium ion. Most carcinogenic nitrosamines have been reported to be mutagenic in one or more laboratories (with considerable interlaboratory variation), and a low percentage of noncarcinogenic nitrosamines are mutagenic. Correlations between mutagenic and carcinogenic potencies were only observed in several studies.

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