Long-Term Stability of Volatile Nitrosamines in Human Urine.

Volatile nitrosamines (VNAs) are established teratogens and carcinogens in animals and classified as probable (group 2A) and possible (group 2B) carcinogens in humans by the IARC. High levels of VNAs have been detected in tobacco products and in both mainstream and sidestream smoke. VNA exposure may lead to lipid peroxidation and oxidative stress (e.g., inflammation), chronic diseases (e.g., diabetes) and neurodegenerative diseases (e.g., Alzheimer's disease). To conduct epidemiological studies on the effects of VNA exposure, short-term and long-term stabilities of VNAs in the urine matrix are needed. In this report, the stability of six VNAs (N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopiperidine, N-nitrosopyrrolidine and N-nitrosomorpholine) in human urine is analyzed for the first time using in vitro blank urine pools fortified with a standard mixture of all six VNAs. Over a 24-day period, analytes were monitored in samples stored at ∼20°C (collection temperature), 4-10°C (transit temperature) and -20 and -70°C (long-term storage temperatures). All six analytes were stable for 24 days at all temperatures (n = 15). The analytes were then analyzed over a longer time period at -70°C; all analytes were stable for up to 1 year (n = 62). A subset of 44 samples was prepared as a single batch and stored at -20°C, the temperature at which prepared samples are stored. These prepared samples were run in duplicate weekly over 10 weeks, and all six analytes were stable over the entire period (n = 22).

Evolution of research on the DNA adduct chemistry of N-nitrosopyrrolidine and related aldehydes.

This perspective reviews our work on the identification of DNA adducts of N-nitrosopyrrolidine and some related aldehydes. The research began as a focused project to investigate mechanisms of cyclic nitrosamine carcinogenesis but expanded into other areas, as aldehyde metabolites of NPYR were shown to have their own diverse DNA adduct chemistry. A total of 69 structurally distinct DNA adducts were identified, and some of these, found in human tissues, have provided intriguing leads for investigating carcinogenesis mechanisms in humans due to exposure to both endogenous and exogenous agents.

Genotoxicity screening for N-nitroso compounds. Electrochemical and electrochemiluminescent detection of human enzyme-generated DNA damage from N-nitrosopyrrolidine.

We report for the first time voltammetric/electrochemiluminescent sensors applied to predict genotoxicity of N-nitroso compounds bioactivated by human cytochrome P450 enzymes.

Analysis of adducts in hepatic DNA of rats treated with N-nitrosopyrrolidine.

N-Nitrosopyrrolidine (NPYR) is a hepatocarcinogen in rats. It is metabolically activated by cytochrome P450 enzymes in the liver leading to the formation of 4-oxobutanediazohydroxide (4) and related intermediates that react with DNA to form adducts. Because DNA adducts are thought to be critical in carcinogenesis by NPYR, we analyzed hepatic DNA of NPYR-treated rats for several adducts: N2-(tetrahydrofuran-1-yl)dGuo (N2-THF-dGuo, 13), N6-THF-dAdo (14), N4-THF-dCyd (17), and dThd adducts 15 and 16. The rats were treated with NPYR in the drinking water, 600 ppm for 1 week, or 200 ppm for 4 or 13 weeks. Hepatic DNA was isolated, enzymatically hydrolyzed, and analyzed by capillary LC-ESI-MS-SIM, which indicated the presence of adducts 13, 14, and 17. Because these adducts can be unstable at the deoxyribonucleoside level, further analyses were carried out using DNA treated with NaBH3CN, which converts adducts 13-17 to N2-(4-hydroxybut-1-yl)dGuo [N2-(4-HOB)dGuo, 18], N6-(4-HOB)dAdo (19), O2-(4-HOB)dThd (20), O4-(4-HOB)dThd (21), and N4-(4-HOB)dCyd (22). [15N]-Labeled analogues of adducts 18-20 and 22 were synthesized and used in this analysis, which was performed by capillary LC-ESI-MS/MS-SRM. Convincing evidence for the presence of adducts 18-22 was obtained. Levels of 18, 19, 20, and 21 were (mumol/mol dGuo): 3.41-5.39, 0.02-0.04, 2.56-3.87, and 2.28-5.05, respectively. Compound 22 was not quantified due to interfering peaks. These results provide the first evidence for tetrahydrofuranyl-substituted DNA adducts in the livers of rats treated with NPYR. The finding of dAdo and dThd adducts is of particular interest since previous studies have shown that NPYR causes mutations at AT base pairs in DNA of rat liver.

Cytochrome P450 2A-catalyzed metabolic activation of structurally similar carcinogenic nitrosamines: N'-nitrosonornicotine enantiomers, N-nitrosopiperidine, and N-nitrosopyrrolidine.

N'-Nitrosonornicotine (NNN) and N-nitrosopiperidine (NPIP) are potent esophageal and nasal cavity carcinogens in rats and pulmonary carcinogens in mice. N-Nitrosopyrrolidine (NPYR) induces mainly liver tumors in rats and is a weak pulmonary carcinogen in mice. These nitrosamines may be causative agents in human cancer. alpha-Hydroxylation is believed to be the key activation pathway in their carcinogenesis. P450 2As are important enzymes of nitrosamine alpha-hydroxylation. Therefore, a structure-activity relationship study of rat P450 2A3, mouse P450 2A4 and 2A5, and human P450 2A6 and 2A13 was undertaken to compare the catalytic activities of these enzymes for alpha-hydroxylation of (R)-NNN, (S)-NNN, NPIP, and NPYR. Kinetic parameters differed significantly among the P450 2As although their amino acid sequence identities were 83% or greater. For NNN, alpha-hydroxylation can occur at the 2'- or 5'-carbon. P450 2As catalyzed 5'-hydroxylation of (R)- or (S)-NNN with Km values of 0.74-69 microM. All of the P450 2As except P450 2A6 catalyzed (R)-NNN 2'-hydroxylation with Km values of 0.73-66 microM. (S)-NNN 2'-hydroxylation was not observed. Although P450 2A4 and 2A5 differ by only 11 amino acids, they were the least and most efficient catalysts of NNN 5'-hydroxylation, respectively. The catalytic efficiencies (kcat/Km) for (R)-NNN differed by 170-fold whereas there was a 46-fold difference for (S)-NNN. In general, P450 2As catalyzed (R)- and (S)-NNN 5'-hydroxylation with significantly lower Km and higher kcat/Km values than NPIP or NPYR alpha-hydroxylation (p <0.05). Furthermore, P450 2As were better catalysts of NPIP alpha-hydroxylation than NPYR. P450 2A4, 2A5, 2A6, and 2A13 exhibited significantly lower Km and higher kcat/Km values for NPIP than NPYR alpha-hydroxylation (p <0.05), similar to previous reports with P450 2A3. Taken together, these data indicate that critical P450 2A residues determine the catalytic activities of NNN, NPIP, and NPYR alpha-hydroxylation.

Preferential metabolic activation of N-nitrosopiperidine as compared to its structural homologue N-nitrosopyrrolidine by rat nasal mucosal microsomes.

N-Nitrosopiperidine (NPIP) is a potent rat nasal carcinogen whereas N-nitrosopyrrolidine (NPYR), a hepatic carcinogen, is weakly carcinogenic in the nose. NPIP and NPYR may be causative agents in human cancer. P450-catalyzed alpha-hydroxylation is the key activation pathway by which these nitrosamines elicit their carcinogenic effects. We hypothesize that the differences in NPIP and NPYR metabolic activation in the nasal cavity contribute to their differing carcinogenic activities. In this study, the kinetics of tritium-labeled NPIP or NPYR alpha-hydroxylation mediated by Sprague-Dawley rat nasal olfactory or respiratory microsomes were investigated. To compare alpha-hydroxylation rates of the two nitrosamines, tritiated 2-hydroxytetrahydro-2H-pyran and 2-hydroxy-5-methyltetrahydrofuran, the major NPIP alpha-hydroxylation products, and tritiated 2-hydroxytetrahydrofuran, the major NPYR alpha-hydroxylation product, were quantitated by HPLC with UV absorbance and radioflow detection. These microsomes catalyzed the alpha-hydroxylation of NPIP more efficiently than that of NPYR. K(M) values for NPIP were lower as compared to those for NPYR (13.9-34.7 vs 484-7660 muM). Furthermore, catalytic efficiencies (V(max)/K(M)) of NPIP were 20-37-fold higher than those of NPYR. Previous studies showed that P450 2A3, present in the rat nose, also exhibited this difference in catalytic efficiency. For both types of nasal microsomes, coumarin (100 muM), a P450 2A inhibitor, inhibited NPIP and NPYR alpha-hydroxylation from 63.8 to 98.5%. Furthermore, antibodies toward P450 2A6 inhibited nitrosamine alpha-hydroxylation in these microsomes from 68.8 to 78.4% whereas antibodies toward P450 2E1 did not inhibit these reactions. Further immunoinhibition studies suggest some role for P450 2G1 in NPIP metabolism by olfactory microsomes. In conclusion, olfactory and respiratory microsomes from rat nasal mucosa preferentially activate NPIP over NPYR with P450 2A3 likely playing a key role. These results are consistent with local metabolic activation of nitrosamines as a contributing factor in their tissue-specific carcinogenicity.

Comparative metabolism of N-nitrosopiperidine and N-nitrosopyrrolidine by rat liver and esophageal microsomes and cytochrome P450 2A3.

N-nitrosopiperidine (NPIP) is a potent esophageal carcinogen in rats whereas structurally similar N-nitrosopyrrolidine (NPYR) induces liver, but not esophageal tumors. NPIP is a possible causative agent for human esophageal cancer. Our goal is to explain mechanistically these differing carcinogenic activities in the esophagus. We hypothesize that differences in metabolic activation of these nitrosamines could be one factor accounting for their differing carcinogenicity. alpha-Hydroxylation is the key metabolic activation pathway leading to nitrosamine-induced carcinogenesis. In this study, we examined the alpha-hydroxylation rates of [3,4-(3)H]NPIP and [3,4-(3)H]NPYR by male F344 rat esophageal and liver microsomes. The major alpha-hydroxylation products of NPIP and NPYR, 2-hydroxytetrahydro-2H-pyran (2-OH-THP) and 2-hydroxytetrahydrofuran (2-OH-THF), respectively, were monitored by high performance liquid chromatography with radioflow detection. NPIP or NPYR (4 microM) was incubated with varying concentrations of esophageal microsomes and co-factors. Microsomes converted NPIP to 2-OH-THP with a 40-fold higher velocity than NPYR to 2-OH-THF. Similar results were observed in studies with NPIP and NPYR at substrate concentrations between 4 and 100 micro M. Kinetics of NPIP alpha-hydroxylation were biphasic; K(M) values were 312 +/- 50 and 1600 +/- 312 microM. Expressed cytochrome P450 2A3, found in low levels in rat esophagus, was a good catalyst of NPIP alpha-hydroxylation (K(M) = 61.6 +/- 20.5 microM), but a poor catalyst of NPYR alpha-hydroxylation (K(m) = 1198 +/- 308 micro M). Cytochrome P450 2A3 may play a role in the preferential activation of NPIP observed in rat esophagus. Liver microsomes metabolized NPYR to 2-OH-THF (V(max)/K(M) = 3.23 pmol/min/mg/ microM) as efficiently as NPIP to 2-OH-THP (V(max)/K(M) = 3.80-4.61 pmol/min/mg/ microM). We conclude that rat esophageal microsomes activate NPIP but not NPYR whereas rat liver microsomes activate NPIP and NPYR. These results are consistent with previous findings that tissue-specific activation of nitrosamines contributes to tissue-specific tumor formation.

Dietary exposure and urinary excretion of total N-nitroso compounds, nitrosamino acids and volatile nitrosamine in inhabitants of high- and low-risk areas for esophageal cancer in southern China.

We assessed the exposure of total N-nitroso compounds (TNOCs) in the inhabitants of high- and low-risk areas for esophageal cancer in southern China. Samples of 24 hr diet and 12 hr overnight urine were collected from 120 male adults in each of the 2 areas, a high-risk area (Nan'ao County) and a low-risk area (Lufeng County) for esophageal cancer. Annual standardized mortality rates of esophageal cancer in Nan'ao and Lufeng are 110/10(6) and 10/10(6) respectively. The 240 healthy male subjects (35-64 years old) were selected by a 3-stage random cluster sample procedure. Levels of TNOCs, NAAs and volatile nitrosamines in the samples were measured. The TNOC detection rate (95%) in the diet, the TNOC daily intake (4.25 +/- 0.84 micromol), TNOC excretion levels (0.04 +/- 0.01 nmol/12 hr) and daily intake of volatile nitrosamines (5.84 +/- 0.71 micromol) in the high-risk area were significantly greater than values in the low-risk area (A +/- B = mean +/- SE). The TNOC detection rate in the diet, the TNOC daily intake, TNOC excretion levels and daily intake of volatile nitrosamines in the low-risk area were 70%, 0.25 +/- 0.06 micromol, 0.02 +/- 0.01 nmol/12 hr and 3.18 +/- 0.31 micromol, respectively. NAA excretion levels showed no difference between the 2 areas (16.3 +/- 7.18 micromol/12 hr for Nan'ao and 31.2 +/- 26.4 micromol/12 hr for Lufeng). Thus, TNOCs are implicated in the etiology of esophageal cancer in southern China.

Mutagenicity of N-nitrosodiethylamine in the Ames test with S. typhimurium TA1535 is due to volatile metabolites and is not dependent on cytochrome P4502E1 induction.

N-Nitrosodiethylamine (NDEA) is carcinogenic in all investigated animal species at relatively low dosages. No threshold has been detected for these carcinogenic effects. The substance has been extensively investigated in various in vitro systems, revealing only weak mutagenicity at relatively high dosages. We reinvestigated NDEA in the Ames test with Salmonella typhimurium TA1535 to establish appropriate modifications of the standard Ames test protocol, to achieve a dose-dependent mutagenic response at a reasonably low dose range. Two main modifications were evaluated. Since the metabolism of dialkylnitrosamines is postulated to be mainly dependent on cytochrome P4502E1, a pyrazole-induced rat liver S9 was applied. The second modification involved a gastight preincubation, since metabolites of NDEA might evaporate from the incubation mixture. Cytochrome P4502E1 induction in Wistar rats was achieved by pyrazole treatment. For comparison, a rat liver S9-fraction produced by beta-naphtoflavone/phenobarbital induction was used. N-Nitrosopyrrolidine served as positive control for pyrazole-induced S9-mix with TA1535. NDEA showed no mutagenic response under all test conditions in the presence of pyrazole-induced S9-mix. A strong mutagenic response, exceeding the base rate up to 15-fold at a dose range of 25-1000 microg/plate, was observed using beta-naphtoflavone/phenobarbital-induced S9-mix, gastight preincubation and TA1535. In conclusion the Ames test with gastight preincubation can be useful for the testing of volatile compounds or substances leading to gaseous metabolites. The weak response of NDEA in the Ames test observed previously seems mainly to be due to the volatile character of its mutagenic metabolites. Our results do not support the hypothesis that cytochrome P4502E1 is a major toxifying enzyme for the formation of Ames-test-positive metabolites from NDEA.

Reactions of alpha-acetoxy-N-nitrosopyrrolidine and crotonaldehyde with DNA.

alpha-Acetoxy-N-nitrosopyrrolidine (alpha-acetoxyNPYR) is a stable precursor to alpha-hydroxyNPYR, the initial product of metabolism and proposed proximate carcinogen of NPYR. Crotonaldehyde (2-butenal) is a metabolite of NPYR and also a mutagen and carcinogen. Both alpha-acetoxyNPYR and crotonaldehyde are known to form DNA adducts, but these reactions have not been completely characterized. In previous studies, we detected substantial amounts of unidentified radioactivity in hydrolysates of DNA that had been reacted with radiolabelled alpha-acetoxyNPYR. We have now characterized these products as 2-hydroxytetrahydrofuran, the cyclic form of 4-hydroxybutanal, and paraldol, the dimer of 3-hydroxybutanal. They were characterized by comparison with standards and by comparison of their derived 2,4-dinitrophenylhydrazones with standards. [3H]H2O was also identified. 2-Hydroxytetrahydrofuran is the major product in neutral thermal hydrolysates of alpha-acetoxyNPYR-treated DNA and is derived predominantly from N2-(tetrahydrofuran-2-yl)deoxyguanosine 8. Paraldol is present to a lesser extent than 2-hydroxytetrahydrofuran in these reactions and is formed from paraldol-releasing adducts, which in turn are produced by the reaction of crotonaldehyde or paraldol, solvolysis products of alpha-acetoxyNPYR, with DNA. Paraldol is a major product in hydrolysates of crotonaldehyde-treated DNA, being present in amounts 100 times greater than those of previously identified adducts. These results provide a more complete picture of the reactions of alpha-acetoxyNPYR with DNA and yield some new insights on possible endogenous DNA adducts formed from crotonaldehyde.

A cyclic N7,C-8 guanine adduct of N-nitrosopyrrolidine (NPYR): formation in nucleic acids and excretion in the urine of NPYR-treated rats.

N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen that is present in the diet and tobacco smoke and may form endogenously in humans. Biomarkers to assess NPYR exposure and metabolic activation in humans are needed. The cyclic N7,C-8 guanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purin-4(3H)-one (8), which is formed in tissues of rats treated with NPYR, is one potential candidate for such a biomarker. In this study, we evaluated the formation of this and other NPYR adducts in reactions of alpha-acetoxyNPYR with dGuo, Guo, DNA, and RNA and determined the extent of urinary excretion of adduct 8 in rats treated with NPYR. alpha-AcetoxyNPYR, a stable precursor to the major product of NPYR metabolic activation, was allowed to react with dGuo, Guo, DNA, or RNA at 37 degrees C, pH 7. The most striking observation was that the cyclic N7,C-8 guanine adduct 8 was formed 9 times more extensively in the reaction with Guo than with dGuo. It was also formed 2.5 times more extensively in RNA than in DNA. In rats treated with NPYR, levels of the cyclic N7,C-8 guanine adduct 8 were 2 times as high in RNA than in DNA. Rats treated with [14C]adduct 8 excreted 51% of this adduct unchanged in urine. Rats treated with [3,4-3H]NPYR excreted 0.00004% of the dose as adduct 8. The major differences in product formation in reactions of alpha-acetoxyNPYR with dGuo versus Guo are unusual for alkylating agents; potential mechanisms are discussed. The higher levels of adduct 8 in RNA than in DNA suggest that RNA may be superior as a source of adduct 8 as a biomarker.

Supercritical fluid extraction of volatile N-nitrosamines in fried bacon and its drippings: method comparison.

N-Nitrosopyrrolidine (NPYR) and N-nitrosodimethylamine (NDMA), known animal carcinogens, are consistently formed in bacon during frying. As a result, commercial bacon has been subject to regulatory monitoring and compliance for the past 20 years to ensure that N-nitrosamines do not exceed the 10 ppb volatile level. Currently, time-consuming distillation-solvent extraction and solid-phase extraction (SPE) methods are used for this purpose. With an emphasis on reducing solvent use, we investigated supercritical fluid extraction (SFE) using supercritical carbon dioxide (SC-CO2) for isolation of volatile nitrosamines common to fried bacon. Eighteen fried bacon samples were analyzed for NPYR and NDMA by SFE, SPE, mineral oil distillation (MOD), and low-temperature vacuum distillation (LTVD) methods, using the same gas chromatographic-chemiluminescence detection (thermal energy analyzer) conditions. The range of values for SFE was 0.7 to 20.2 ppb for NPYR and none detected (ND) to 2.4 ppb for NDMA. Analysis of variance of the NPYR data showed a significant difference (p < 0.05) between SFE and SPE results and significant differences between these and those obtained by MOD and LTVD. Overall, SFE was superior to the other methods with the highest recoveries, best repeatability, rapidity of analysis, and solvent-sparing characteristics. Similar results were obtained for SFE after comparison with distillation and SPE methods for determining the same nitrosamines in fried bacon drippings.

Formation of 7-(4-oxobutyl)guanine in hepatic DNA of rats treated with N-nitrosopyrrolidine.

We have reported previously the formation of two structurally distinct exocyclic guanine adducts (adducts 1 and 6) in liver DNA of F344 rats treated with N-nitrosopyrrolidine (NPYR). In this study, we detected and characterized a previously unidentified guanine adduct in liver DNA of NPYR-treated rats. The structure of this adduct was established as 7-(4-oxobutyl)guanine (adduct 2) by comparison with the synthetic standard and confirmed by NaBH4 reduction to 7-(4-hydroxybutyl)guanine. The level of adduct 2 in liver DNA of F344 rats treated with 450 mg/kg of NPYR by i.p. administration was 643 +/- 9 mumol/mol guanine, approximately one-third of the level of adduct 1. This study is the first to demonstrate the in vivo formation of a formylalkyl-substituted guanine adduct by a nitrosamine.

Formation and persistence of a DNA adduct in rodents treated with N-nitrosopyrrolidine.

The rate of formation and the persistence of an exocyclic guanine adduct formed in DNA of rodents treated with various doses of N-nitrosopyrrolidine (NPYR) have been determined. NPYR is hepatocarcinogenic to the rat and forms a covalent adduct in liver DNA; this adduct was recently identified as 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2, 1-f]purine-4[3H]-one. Dose-dependent amounts of adduct formed in liver, kidney and lung DNA of rats, hamsters and mice given oral doses (56-900 mg/kg body wt) of NPYR. The persistence of the adduct in DNA after administration of low doses of NPYR to rats was greatest in the target organ, i.e. the liver; at high doses of NPYR, adduct levels in DNA changed little over a period of at least 72 h. In the hamster, in which NPYR is carcinogenic to the lung but apparently not the liver, the adduct level in liver DNA was an order of magnitude greater than in lung or kidney DNA for a dose of NPYR of 225 or 900 mg/kg body wt; persistence of the adduct in lung DNA was only slightly longer than in liver DNA. The formation and persistence of the 7,8-pyridoguanine adduct in the rat appeared to be consistent with the organotropy of this carcinogen, but this was not true for the hamster, a species that seems to be more resistant to induction of liver and kidney cancer by this carcinogen. Imidazole, an inhibitor of microsomal amine oxidase, and disulfiram, an inhibitor of aldehyde dehydrogenase, decreased metabolic activation of NPYR to an alkylating intermediate; inducers and inhibitors of cytochrome P450 monooxygenases had little effect on the metabolic activation of NPYR to an alkylating agent.

Detection of cyclic 1,N2-propanodeoxyguanosine adducts in DNA of rats treated with N-nitrosopyrrolidine and mice treated with crotonaldehyde.

Cyclic 1,N2-propanodeoxyguanosine adducts are formed in vitro in DNA treated with alpha-acetoxy-N-nitrosopyrrolidine or its metabolite, crotonaldehyde. However, the in vivo formation of these cyclic adducts in DNA has not been demonstrated due to the lack of a sensitive detection method. In this study, a 32P-postlabeling method specific for the detection of 1,N2-propanodeoxyguanosine adducts was developed by using the corresponding 3'-monophosphates as standards. This method was validated by using DNA modified in vitro. It was then applied for the in vivo experiments in which hepatic DNA of rats treated with N-nitosopyrrolidine (NPYR) (total dose, 1.0 mmol) in drinking water or skin DNA of Sencar mice treated topically with crotonaldehyde (1.4 mmol) was isolated and subjected to 32P-postlabeling analysis. 1,N2-Propanodeoxyguanosine adducts were detected in these DNA samples. The minimal levels of adducts from liver DNA and skin DNA detected were estimated to be approximately 0.06 and approximately 0.24 mumol/mol guanine respectively. Interestingly, a background adduct spot chromatographically indistinguishable from the 1,N2-cyclic adducts was observed in the liver DNA of untreated rats. However, no such background adduct was detected in skin DNA of mice. This method demonstrated for the first time the in vivo formation of the cyclic 1,N2-propanodeoxyguanosine adducts.

Detection of exocyclic guanine adducts in hydrolysates of hepatic DNA of rats treated with N-nitrosopyrrolidine and in calf thymus DNA reacted with alpha-acetoxy-N-nitrosopyrrolidine.

This report describes the isolation and characterization of DNA adducts formed in vitro from alpha-acetoxy-N-nitrosopyrrolidine and in rats treated with the hepatocarcinogen N-nitrosopyrrolidine. Esterase-catalyzed hydrolysis of alpha-acetoxy-N-nitrosopyrrolidine in the presence of calf thymus DNA, followed by neutral thermal hydrolysis of the DNA, resulted in formation of three previously unknown Adducts 1-3. They were isolated and characterized by their UV, mass, and proton magnetic resonance spectra as the exocyclic 7,8-guanine adducts 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purine-4(3H)-one (Adduct 1), and cis- and trans-2-amino-7,8-dihydro-8-hydroxy-6-methyl-3H-pyrrolo[2,1-f] purine-4(6H)-one (Adducts 2 and 3). Adduct 1 was formed by addition of 4-oxobutyl diazohydroxide, or a related carbonium ion, to the 7 and 8 positions of guanine. Adducts 2 and 3 resulted from Michael addition of 2-butenal to the 7 and 8 positions of guanine. Esterase-catalyzed hydrolysis of alpha-acetoxy-N-nitrosopyrrolidine in the presence of DNA also produced the exocyclic 1,N2-propanodeoxyguanosine Adducts 4a and 4b which we have previously described. Neutral thermal hydrolysates of hepatic DNA isolated from rats treated with N-nitrosopyrrolidine contained a fluorescent adduct, as previously reported (E.J. Hunt and R.C. Shank, Biochem. Biophys. Res. Commun., 104: 1343, 1982). This fluorescent adduct was shown to be identical to Adduct 1. Adducts 2, 3, 4a, and 4b were not detected in hepatic DNA hydrolysates from these animals. The results of this study provide the first example of a structurally characterized DNA adduct formed in vivo from a cyclic nitrosamine and support the alpha-hydroxylation hypothesis of cyclic nitrosamine metabolic activation.

DNA binding by [2,5-14C]N-nitrosopyrrolidine in excision-repair proficient and deficient strains of Salmonella. Evidence for a major premutagenic adduct.

Little is known about the nature and possible genotoxic effects of the DNA adducts formed by N-nitrosopyrrolidine (NPYR) in whole animals. DNA binding in DNA isolated from [2,5-14C]NPYR-treated Salmonella was studied and attempts were made to monitor DNA adducts and correlate DNA binding with mutagenesis. NPYR was metabolized by hamster liver S-9 fraction in the presence of S.typhimurium TA1535 (uvrB-) or TA1975(uvrB+). DNA isolated from TA1535 contained about three times as much radioactivity as that isolated from TA1975, and NPYR-induced mutagenesis was several-fold higher in TA1535. The fraction of radioactivity incorporated into TA1535 was approximately 10(-5). Thermal hydrolysis of the 14C-containing DNA at neutral pH, followed by precipitation, released approximately 2/3 of the radioactivity into the supernatant. HPLC analysis of the supernatant revealed one major peak. This peak was absent in DNA from TA1975. Acid hydrolysis of the DNA precipitate after neutral hydrolysis released most of the residual radioactivity. Several small peaks were observed after HPLC analysis of the TA1535 acid hydrolysate or the TA1975 acid hydrolysate. These results demonstrate that NPYR is capable of binding to Salmonella DNA yielding one major product after hydrolysis and this DNA binding product appears to be repaired by the excision repair system. The fact that the major peak of radioactivity released from Salmonella is only found in the strain which is efficiently reverted by NPYR suggests that mutagenesis is dependent on the DNA modification leading to this peak.

Reconstitution of rabbit liver microsomal N-nitrosopyrrolidine alpha-hydroxylase activity.

The in vitro alpha-hydroxylation of N-nitrosopyrrolidine (NPYR) by both isolated rabbit liver microsomes and purified cytochrome P-450 isozymes was investigated. Microsomes from untreated rabbits catalyzed the alpha-hydroxylation of NPYR at rates similar to those reported previously for rats, mice, and hamsters. The effect of established inducers of microsomal P-450 caused complex changes in apparent rates of alpha-hydroxylation of NPYR which made interpretation of responses to inducer pretreatment difficult and suggested the participation of multiple cytochrome P-450 isozymes in the metabolism of NPYR. Partial inhibition of alpha-hydroxylase activity by antibodies against rabbit isozymes 2, 3a, and 5 indicated the participation of at least these three isozymes in microsomal catalysis. Reconstitution studies using purified rabbit isozymes 2, 3a, 3b, 3c, 4, and 6 indicated that isozymes 2, 3a, 4, and 6 possessed significant alpha-hydroxylase activity with isozymes 3a and 6 exhibiting the highest activity when assayed at 20 mM NPYR. As NPYR concentrations were decreased, the rates of catalysis for the reconstituted systems were differentially decreased such that isozyme 3a exhibited the highest activity at low NPYR concentrations. These data indicate that isozyme 3a is the preferred catalyst for the alpha-hydroxylation of NPYR at low substrate concentrations and suggest that conditions such as chronic ethanol consumption which lead to the induction of isozyme 3a in rabbits or its orthologue in other species can account for enhanced rates of alpha-hydroxylation and metabolic activation of NPYR.

Identification of crotonaldehyde as a hepatic microsomal metabolite formed by alpha-hydroxylation of the carcinogen N-nitrosopyrrolidine.

Crotonaldehyde (2-butenal), which reacts with DNA and is mutagenic and carcinogenic, was identified as a hepatic microsomal metabolite of the hepatocarcinogen N-nitrosopyrrolidine. Incubation mixtures of N-nitrosopyrrolidine, cofactors, and hepatic microsomes from Aroclor pretreated or control F344 rats were derivatized with (2,4-dinitrophenyl)hydrazine reagent and the resulting mixtures analyzed by high-performance liquid chromatography. Crotonaldehyde (2,4-dinitrophenyl)hydrazone was identified by its retention time in two different systems and by its ultraviolet and mass spectrum. The ratio of 4-hydroxybutyraldehyde, which has previously been identified as a metabolite of NPYR, to crotonaldehyde was 1.5-2 over a range of substrate concentrations. The approximate values of Km and nu max for crotonaldehyde were 5.8 mM and 0.6 nmol/min/mg of protein and for 4-hydroxybutyraldehyde 14.1 mM and 1.7 nmol/min/mg of protein, for substrate concentrations between 1 and 8 mM, with microsomes from Aroclor pretreated rats. The ratio of 4-hydroxybutyraldehyde to crotonaldehyde was 1.9 upon esterase-catalyzed solvolysis of alpha-acetoxy-N-nitrosopyrrolidine, a stable precursor to the initial product of N-nitrosopyrrolidine alpha-hydroxylation. These results demonstrate that crotonaldehyde is formed upon metabolic alpha-hydroxylation of N-nitrosopyrrolidine and suggest that it may be involved in N-nitrosopyrrolidine-macromolecule interactions.

Differing effects of chronic ethanol consumption by mice on liver microsomal metabolism of xenobiotics: 1-nitropyrene, nicotine, aniline, and N-nitrosopyrrolidine.

The effect of ethanol consumption by male CF-1 mice on liver microsomal enzyme activities has been investigated. The total microsomal cytochrome P-450 content was increased by 38%, while cytochrome b5 was decreased by 31%, which are characteristic alterations in liver microsomes following ethanol consumption. Other alterations included a decreased NADPH cytochrome c reductase activity and increased NADPH-supported rates of N-nitrosopyrrolidine and aniline hydroxylation. While ethanol consumption did not alter the total metabolism of nicotine, the rates of N- and C-hydroxylation were differently affected. The 5'-hydroxylation of nicotine was increased by 83%, while the N'-oxidation was decreased by 31%. Changes in the microsomal metabolism of the environmental carcinogen 1-nitropyrene included a slight reduction in the overall metabolism, which can be accounted for by a reduction in the formation of one phenolic metabolite, 1-nitropyren-3-ol.