Probing the Mechanism of the Carcinogenic Activation of <i>N</i>-Nitrosodiethanolamine with Deuterium Isotope Effects:  In Vivo Induction of DNA Single-Strand Breaks and Related in Vitro Assays

Loeppky, Richard N.; Fuchs, Anne; Janzowski, C.; Humberd, Christina; Goelzer, Petra; Schneider, Heiko; Eisenbrand, Gerhard

doi:10.1021/tx9801716

Cited by 22 publications

(31 citation statements)

References 41 publications

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“…Deuterium isotope effects have been used extensively in the study of the mechanisms of nitrosamine-induced carcinogenesis (18,(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)(81)(82). The first experiment to utilize deuterium substitution in the study of nitrosamineinduced cancer demonstrated that perdeuteration of NDMA lowered the liver carcinogenicity of this compound in rats (65).…”

Section: (4r)-[4-2 H 1 ]Nnk Was Less Tumorigenic Than Nnk and (4s)-[4mentioning

confidence: 99%

Stereospecific Deuterium Substitution Attenuates the Tumorigenicity and Metabolism of the Tobacco-Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Jalas

McIntee

Kenney

et al. 2003

Chem. Res. Toxicol.

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Stereochemical determinants of the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were investigated using the stereospecifically deuterated isotopomers (4R)-[4-(2)H(1)]NNK and (4S)-[4-(2)H(1)]NNK. Upon ip administration to groups of 20 female A/J mice, NNK and (4S)-[4-(2)H(1)]NNK exhibited similar lung tumorigenicity at three different doses, whereas (4R)-[4-(2)H(1)]NNK was 2-fold less tumorigenic at all three doses. In a parallel experiment, levels of O(6)-methylguanine and 7-methylguanine were 2-fold lower in lung DNA of mice treated with (4R)-[4-(2)H(1)]NNK than in mice treated with NNK or (4S)-[4-(2)H(1)]NNK. To corroborate these in vivo data, the in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda (Sf9)-expressed mouse cytochrome p450s 2A4 and 2A5. Kinetic isotope effects on the apparent V(max) ((D)V) for the product of NNK 4-hydroxylation, OPB, were 2.7 +/- 0.2 and 2.8 +/- 0.4 when (4R)- and (4S)-[4-(2)H(1)]NNK were incubated with mouse lung microsomes, respectively. The (D)V values for OPB formation were 3.2 +/- 0.2 and 2.2 +/- 0.2 when (4R)-[4-(2)H(1)]NNK was the substrate for p2A4 and 2A5, respectively, whereas they were 1.3 +/- 0.1 and 1.1 +/- 0.1 when (4S)-[4-(2)H(1)]NNK was the substrate for these respective enzymes. Analysis of an OPB derivative (10) for deuterium content by LC/MS confirmed the results from the kinetic assays and indicated that p450s 2A4 and 2A5 preferentially abstract the pro-R 4-hydrogen of NNK. The results obtained using Sf9-expressed p450s provide a rationale for the differences observed in the lung tumor and DNA adduct experiments, namely, that the attenuated tumorigenicity of (4R)-[4-(2)H(1)]NNK relative to (4S)-[4-(2)H(1)]NNK is due to prochiral selectivity during p450-catalyzed metabolic activation.

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Section: (4r)-[4-2 H 1 ]Nnk Was Less Tumorigenic Than Nnk and (4s)-[4mentioning

confidence: 99%

Stereospecific Deuterium Substitution Attenuates the Tumorigenicity and Metabolism of the Tobacco-Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Jalas

McIntee

Kenney

et al. 2003

Chem. Res. Toxicol.

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“…Because our deuteration experiments (20) with NDELA in vivo pointed toward an R-hydroxylation pathway for this compound, we reconsidered the applicability of the method shown in Scheme 1 to the detection of glycolaldehyde, a very polar aldehyde, which is also susceptible to osazone reactions with hydrazines to give hydrazones of glyoxal. Isooctane is a very nonpolar organic solvent, and the possibility that it would not extract either the DNP of glycolaldehyde or glyoxal was considered prior to the initiation of the work presented here.…”

Section: Introductionmentioning

confidence: 99%

Microsome-Mediated Oxidation of N-Nitrosodiethanolamine (NDELA), a Bident Carcinogen

Loeppky

Goelzer

2002

Chem. Res. Toxicol.

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N-Nitrosodiethanolamine (NDELA), an environmentally prevalent, potent carcinogen, undergoes competitive rat liver microsome-mediated oxidation at both the alpha (adjacent to N)- and beta-positions of the 2-hydroxyethyl chains. The former process, alpha-hydroxylation, is detected by the formation of glycolaldehyde (determined as its 2,4-dinitrophenylhydrazone DNP) that is assumed to arise from the decomposition of the corresponding alpha-hydroxynitrosamine, which is also the progenitor of the 2-hydroxyethyldiazonium ion. This finding refutes prior published work that states that the alpha-hydroxylation of NDELA does not occur. Competitive microsomal oxidation at the beta-position gives the hemiacetal N-nitroso-2-hydroxymorpholine (NHMOR) at a rate 1.5 times alpha-hydroxylation. Glycolaldehyde is oxidized in this system to glyoxal at a rate 39 times the conversion of NDELA to glycolaldehyde. The alpha-hydroxylation of NHMOR at either C-3 or C-5 to give glyoxal or glycolaldehyde, respectively, occurs at respective rates 3-6 times that of the alpha-hydroxylation of NDELA. Ethylene glycol, a hydrolysis product of the 2-hydroxyethyldiazonium ion is shown to undergo microsome mediate oxidation to glyoxal. Ethyl-2-hydroxyethylnitrosamine (NEELA) undergoes a similar set of microsome-mediated oxidations at alpha-position of the ethyl (fastest) and 2-hydroxyethyl groups, as well as beta-oxidation of the 2-hydroxyethyl group, a process which is slightly more rapid than alpha-hydroxylation of the same chain. Comparisons of oxidations rates of these substrates, as manipulated by preinducers, isoniazid, streptozocin, and phenobarbital, and enzyme inhibitors diethyldithiocarbamate and 4-methylpyrazole, with that of dimethylnitrosamine, a substrate for cytochrome P450 2E1, strongly suggest that this isozyme is also responsible for the oxidations reported here. alpha-Deuteration of NDELA practically eliminates its alpha-hydroxylation by microsomes from isoniazid induced rats, but doubles beta-oxidation, while beta-deuteration of this substrate significantly reduces beta-oxidation and enhances alpha-hydroxylation. Since both glyoxal-guanine and 2-hydroxyethyl-DNA base adducts are known to arise from the in vivo administration of NDELA and because this work demonstrates that these two fragments can come from the microsomal oxidation of a single nitrosamine molecule containing the 2-hydroxyethyl group, NDELA and related nitrosamines are bident (two-toothed) carcinogens, a process which is likely to enhance their carcinogenic potency.

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“…The cells were grown at 37°C in a humidified atmosphere containing 5% CO 2 . References for enzymes primarily involved in metabolic activation: [13][14][15] 4 Methylhydroxylation activity of tolbutamide. 5 4' -Hydroxylation activity of S-mephenytoin.…”

Section: Introductionmentioning

confidence: 99%

Detection of in vitro genotoxicity of pro-mutagens using the comet assay under human and rat liver S9 fractions

Kawaguchi

Nakamura

Tsuda

et al. 2018

MOJT

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The metabolic activating ability of human liver S9, human lung S9 (HLuSP-E), and rat liver S9 were compared. One of the human liver S9 (HLSP-E) was prepared from a pool from 15 donors and the other (HLS-014E) from a liver sample that had high levels of P450 activities. Rat liver S9 was prepared from male Sprague-Dawley rats either untreated (RLSP-E) or pretreated with phenobarbital/5,6-benzoflavone (RL-PB/BF). Human lymphoma WTK1 cells were treated with 37 pro-mutagens that required cytochrome P450 or NADPH-P450 reductase for metabolic activation and their genotoxicity was determined by the comet assay. HLS-014E and RL-PB/BF had a higher P450 enzyme activity and activated a greater variety of pro-mutagens than others. The results of the human liver S9 and RL-PB/BF were discrepant for only 2 out of 37 pro-mutagens; phenacetin was positive with human liver S9 but not with RL-PB/BF, and benz[a]anthrathene was positive with RL-PB/BF but not with human liver S9. Both RL-PB/BF and human liver S9 activated pyrene, IQ, MeIQ, 1-nitropyrene, and N-nitrosopiperidine while RLSP-E did not. In spite of the existence of species differences in CYP isoforms and differences of CYP activity, therefore, there were no appreciable qualitative differences in pro-mutagen activating ability of the human liver S9 and RL-PB/BF, suggesting the possibility that the genotoxicity of some pro-mutagens that are genotoxic with human liver S9 are missed by the use of liver S9 from rats that were not pretreated to induce CYP. Therefore, conventionally used CYP-induced rat liver S9 fraction is useful for the evaluation of the genotoxic risk for humans in spite of the species difference in CYP isoforms.Keywords: rat liver S9, human liver S9, metabolic activation, comet assay MOJ Toxicology Research ArticleOpen Access Detection of in vitro genotoxicity of pro-mutagens using the comet assay under human and rat liver S9 fractions 256

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Probing the Mechanism of the Carcinogenic Activation of N-Nitrosodiethanolamine with Deuterium Isotope Effects: In Vivo Induction of DNA Single-Strand Breaks and Related in Vitro Assays

Cited by 22 publications

References 41 publications

Stereospecific Deuterium Substitution Attenuates the Tumorigenicity and Metabolism of the Tobacco-Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Stereospecific Deuterium Substitution Attenuates the Tumorigenicity and Metabolism of the Tobacco-Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Microsome-Mediated Oxidation of N-Nitrosodiethanolamine (NDELA), a Bident Carcinogen

Detection of in vitro genotoxicity of pro-mutagens using the comet assay under human and rat liver S9 fractions

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