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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)., (Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

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.

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

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.

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.

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.

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.

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., (Copyright 2002 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

This report represents a study of the total metabolism of the hepatocellular carcinogen, N-nitrosopyrrolidine (NO-PYR), by rat liver microsomes and postmicrosomal supernatant. [2,5-14C]NO-PYR, which is totally extractable from aqueous solution with methylene chloride, is converted to radioactive nonmethylene chloride-extractable products by these fractions. The initial rate of conversion to nonmethylene chloride-extractable products follows simple Michaelis-Menten kinetics with an apparent Km of 3.6 x 10(-4) M NO-PYR. The major products of NO-PYR metabolism by rat liver microsomes and postmicrosomal supernatant have been isolated and identified. One product of metabolism of NO-PYR is 2-hydroxytetrahydrofuran formed by alpha-hydroxylation by the microsomes. In the presence of postmicrosomal supernatant enzymes, this compound exists only as a transient intermediate which is rapidly converted to 1,4-butanediol or gamma-hydroxybutyrate. These compounds may be cycled into general cellular metabolism resulting in the production of CO2. Two minor pathways of metabolism have also been found.

Tissues derived from male Sprague-Dawley rats which had been pair-fed isocaloric ethanol or carbohydrate control diets for 4 weeks were used to determine the effect of chronic ethanol exposure on the activation of the smoking and diet-associated procarcinogen nitrosopyrrolidine (NPY). Microsomal preparations from lungs and liver and S9 fractions from alimentary tract mucosa were tested for their capacity to activate NPY to a mutagen by the Ames Salmonella assay. Chronic ethanol exposure resulted in an increased capacity to activate NPY by microsomes derived from liver, lungs, and esophagus but not from stomach. The three tissues in which enhanced activation of NPY was observed in the present study also have been noted in epidemiological studies to be target sites of alcohol abuse-associated increased cancer risk. The results presented here suggest that dietary ethanol's effect on carcinogen metabolism may be responsible, at least in part, for the increased cancer risk observed in chronic alcohol abusers.

The metabolism of N-nitrosopyrrolidine (NPYR) to 4-hydroxybutanal (4-HB) (the first stable product of the putative alpha-hydroxylation pathway) and its bioactivation in the Ames bacterial mutagenicity test system were examined in the presence of a number of inhibitors. Both SKF 525A and piperonyl butoxide were found to be potent inhibitors of the production of 4-HB by rat liver microsomal preparations but were ineffective in the mutagenicity model with liver S-9 from either untreated or Aroclor 1254 pretreated rats. In addition two inhibitors of the mutagenic activity of N-nitrosodimethylamine (NDMA) in this system, 2-phenylethylamine and benzimidazole failed to reduce the activity of NPYR. These results suggest that the bioactivation of NPYR may proceed by processes other than the cytochrome P-450 dependent route generating 4-HB and the amine oxidase catalysed route implicated in NDMA activation. Evidence was also obtained of a second cytosol dependent bioactivation step involving a microsome generated pre-mutagen. This activity may be responsible, at least in part, for the enhancement by cytosol of the mutagen producing activity of liver microsomes from Aroclor 1254 pretreated (but not control) rats.

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.

A high-pressure liquid chromatographic assay for alpha hydroxylation of N-nitrosopyrrolidine by isolated hepatic microsomes was developed. Mixtures consisting of N-nitrosopyrrolidine, microsomes, and an NADPH-generating system were incubated at 37 degrees. The major product of alpha hydroxylation of N-nitrosopyrrolidine, 2-hydroxytetrahydrofuran, was trapped by the addition of 2,4-dinitrophenylhydrazine reagent to form 4-hydroxybutyraldehyde-2,4-dinitrophenylhydrazone. The latter was quantified by reverse-phase high-pressure liquid chromatography. Under optimal conditions, as determined by varying protein and substrate concentrations, the alpha hydroxylation of N-nitrosopyrrolidine was linear for at least 90 min and showed characteristics typical of the microsomal mixed-function oxidase system, such as inhibition by CO and induction by pretreatment of male F-344 rats with Aroclor. N-Nitrosopyrrolidine exhibited type II spectral changes upon interaction with isolated hepatic microsomes. A close correspondence between binding affinity and alpha hydroxylation of N-nitrosopyrrolidine was observed.

Liver DNA and RNA were isolated from rats treated with the liver carcinogens N-nitrosopyrrolidine (NPYR) and N-nitrosohexamethyleneimine (NHX). After hydrolysis in 70% perchloric acid (100 degrees C, 1.0 h), 70% of the radioactivity in both the DNA and RNA hydrolysates chromatographed as a single peak. The material from both hydrolysates had comparable Rf values on cation exchange and Sephadex G-10 chromatography. Subsequent experiments indicated this material was volatile. After depurination (0.1 M HCl) of the DNA from NPYR- and NHX-treated rats, Sephadex G-10 chromatography separated only a single radioactive peak which co-eluted with the apurinic acid at the void volume. The material which comprised this peak was not volatile. After dialysis of the same 0.1 M HCl hydrolysate from NHX-treated rats, 98% of the radioactivity remained attached to the apurinic acid. These 2 cyclic nitrosamines appear to produce alkylating species that: (1) are capable of extensive, if not exclusive, phosphotriester formation; or (2) have 2 active sites that cross-link to keep purines attached to apurinic acid after 0.1 M HCl hydrolysis.

N-Nitrosopyrrolidine is carcinogenic for the liver but not for the lung. Optimal conditions for the metabolism of N-nitrosopyrrolidine (NO-PYR) by rat lung microsomes and post-microsomal supernatant were determined. Neither lung nor liver subcellular fractions were able to form detectable amounts of 3-hydroxyNO-PYR. Liver and lung microsomes could only alpha-hydroxylate NO-PYR, and even though the rates of lung reactions were considerably slower than those of liver, similar products were produced by the action of both lung and liver supernatants on microsomal products. The apparent Km for the lung microsome plus supernatant reaction was approx. 20 mM as compared with 0.36 mM for the liver system. Since both the target and non-target tissue extracts could metabolize NO-PYR by the same pathways, we speculate that alpha-hydroxylation of NO-PYR may be a necessary, but not sufficient cause for its carcinogenic actions.

Male F344 rats were pretreated with various dietary compounds, and the effects of pretreatment on the in vitro alpha-hydroxylation of N-nitrosopyrrolidine or N'-nitrosonornicotine were determined in assays with liver microsomes or cultured esophagus, respectively. Dietary compounds included phenols, cinnamic acids, coumarins, indoles, and isothiocyanates. Pretreatments were carried out either by administering the compound by gavage 2 hr prior to sacrifice (acute protocol) or by adding the compound to the diet for 2 weeks (chronic protocol). Acute pretreatment with benzyl isothiocyanate, allyl isothiocyanate, phenethyl isothiocyanate, phenyl isocyanate, and benzyl thiocyanate but not sodium thiocyanate inhibited formation of alpha-hydroxylation products of both nitrosamines. When the chronic pretreatment protocol was used, only phenyl isothiocyanate and sodium thiocyanate inhibited formation of alpha-hydroxylation products of both nitrosamines. Pretreatments with butylated hydroxyanisole, p-methoxyphenol, or N-acetylcysteine had little, if any, effect on the alpha-hydroxylation of N-nitrosopyrrolidine or N'-nitrosonornicotine. Chronic pretreatment with p-hydroxycinnamic acid, 4-hydroxy-3- methoxycinnamic acid, coumarin, umbelliferone, limetine , indole, indole-3-carbinol, indole-3-acetonitrile, and L-tryptophan induced activity for the alpha-hydroxylation of N-nitrosopyrrolidine. The results of this study indicate that isothiocyanates are possible candidates for further study as potential inhibitors of carcinogenesis by N-nitrosopyrrolidine and N'-nitrosonornicotine.

High-pressure liquid chromatographic methods were developed to study microsomal alpha-hydroxylation, which is likely to be an activation mechanism for N-nitrosopyrrolidine (NPYR) and N'-nitrosonornicotine (NNN). The effects of ethanol, phenobarbital and 3-methylcholanthrene pretreatment on the alpha-hydroxylation of NPYR and NNN were determined using F-344 rats and Syrian golden hamsters. In the rat, 3-methylcholanthrene pretreatment decreased the rates of alpha-hydroxylation of NPYR and 5'-hydroxylation of NNN, but increased the rate of 2'-hydroxylation of NNN. Phenobarbital pretreatment of rats did not markedly affect rates of alpha-hydroxylation of NPYR or NNN. In the hamster, 3-methylcholanthrene pretreatment increased the rate of alpha-hydroxylation of NPYR, but did not affect the rates of alpha-hydroxylation of NNN. Phenobarbital or ethanol pretreatment of hamsters resulted in increased rates of alpha-hydroxylation of NPYR and 5'-hydroxylation of NNN. The induction of alpha-hydroxylation of NNN and NPYR by ethanol suggests that these nitrosamines may be more carcinogenic in alcohol-treated animals.

Nitrosopyrrolidine (NO-PYR), an hepatocellular carcinogen, is rapidly metabolized to CO2 by hepatocytes freshly isolated from the livers of male Fischer rats. Using CO2 evolution as a measure of NO-PYR metabolism, we observed two kinetic constants; a high affinity component (Km = 0.11 mM), and a lower affinity component (K m = 3.2 mM). The high affinity component has similar kinetic constants to those observed for in vitro reactions with microsomes plus cytosol (Km = 0.36 mM). Therefore, it is probable that the microsomal reaction is the limiting factor in the metabolism of NO-PYR in hepatocytes. NO-PYR may be metabolized to CO2 through normal anaplerotic sequences. Some metabolites of NO-PYR which have been tentatively identified are gamma-hydroxybutyrate, succinic semialdehyde, 3,4-dihydroxybutyric acid lactone, lactate, acetate, pyruvate, glyoxylate, gamma-aminobutyrate and alanine. 2-Hydroxytetrahydrofuran (2-hydroxy-THF). a product of alpha-hydroxylation was detected at low levels in only one of four reactions. 3-Hydroxy-NO-PYR is present but represents only a small percentage of the total metabolism and is probably of little significance in the overall catabolism of NO-PYR in hepatocytes.

Female A/J mice were maintained on NIH-07 diet or on NIH-07 diet containing butylated hydroxyanisole (BHA, a mixture of 2- and 3-tert-butyl-4-hydroxyanisole), 5 mg/g, for 1 week prior to and during 10 weeks of treatment with N-nitrosodimethylamine (NDMA) or N-nitrosopyrrolidine (NPYR), administered in the drinking water. Twenty weeks after nitrosamine treatment ended, mice were sacrificed and lung adenomas were counted. BHA inhibited NDMA tumorigenesis but enhanced NPYR tumorigenesis. Treatment of A/J mice for three weeks with BHA (5 mg/g) added to semisynthetic diet increased whole lung microsomal alpha-hydroxylation of NDMA and NPYR, as measured by aldehyde production, and increased lung and hepatic glutathione-S-transferase activities. No evidence was found for formation of S-methylglutathione in incubations with NDMA and hepatic supernatants obtained from BHA treated or control mice. Four h after gavage of NDMA, levels of 7-methylguanine in the lung DNA of mice treated with BHA were higher than in the lung DNA of control mice, but levels of O6-methylguanine in the two groups were the same. The results of this study indicate that BHA treatment increases the microsomal metabolic alpha hydroxylation of both NDMA and NPYR, but has differential effects on their tumorigenic activities.

Three homologous cyclic nitrosamines, nitrosopyrrolidine, nitrosopiperidine and nitrosohexamethyleneimine, were administered to groups of 20 female F344 rats in drinking water, as 0.9 mM solutions. Treatment with the latter 2 compounds lasted 28 weeks, at which time several animals had died with esophageal tumors. All of the animals in these 2 groups were dead by the 36th week of the experiment. Most of the animals given nitrosohexamethyleneimine also had angiosarcomas of the liver, and a few hepatocellular carcinomas were seen. Treatment with nitrosopyrrolidine lasted 50 weeks and the first animal in the group died at the 63rd week with an hepatocellular carcinoma. By the 110th week, almost all of these animals had died with the same type of tumor, and the 2 survivors were then killed. Many of the hepatocellular carcinomas had metastasized. There were no tumors of the liver in the rats treated with nitrosopiperidine.

6 nitrosamines, 5 of which cause rat nasal cancer, were tested for mutagenicity in the TA100 strain of S. typhimurium with rat and rabbit nasal, lung and liver S9 homogenates. The TA98 strain also was used with rabbit tissue homogenates. The two cyclic nitrosamines tested, N-nitrosopiperidine and N-nitrosopyrrolidine, were substantially mutagenic with all rabbit tissue homogenates in TA100, but in the rat only nasal homogenate was effective in activating them. N-Nitrosodi(n)propylamine also was activated by rat nasal tissue homogenate but not by the other rat or rabbit tissue homogenates. Diethanolnitrosamine was a direct mutagen in both TA100 and TA98. N-Nitrosodimethylamine and N-nitrosodiethylamine were not mutagenic under any test conditions. The results indicate that some nitrosamines that cause nasal cancer can be activated by nasal enzymes and that possibly important differences in activating capabilities occur among respiratory tract and hepatic tissues and among animal species.

The environmental carcinogens N-nitrosopyrrolidine (NPYR) and N'-nitrosonornicotine (NNN) were incubated with human liver microsomes to test for the presence of enzymes catalyzing alpha-hydroxylation, which is likely to be an activation mechanism for these compounds. Both nitrosamines underwent alpha-hydroxylation; rates were higher for NPYR than for NNN, as observed in rats. The results indicate that both NPYR and NNN can be metabolically activated by human liver microsomes.

1. By the use of whole-body autoradiography, the localization of N-nitroso[14C]pyrrolidine and its metabolites was demonstrated in the tissues of C57B1-mice and Sprague-Dawley rats. 2. On the basis of the autoradiographic data, tissues were selected and tested for their ability to metabolize N-nitroso[14C]pyrrolidinie to 14CO2 and tissue-bound metabolites. 3. The results indicated that only few tissues had a marked ability to degrade N-nitrosopyrrolidine. These were the liver and the nasal mucosa in both species, the tracheobronchial mucosa in mice, and to a lower extent the kidneys in both species.

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.

Microsomes and postmicrosomal supernatant were prepared from the esophagus and non-grandular stomach of rats. Using these fractions, we could not demonstrate in vitro metabolism of 2,6-dimethyldinitrosopiperazine (DMDNP), a potent esophageal and non-grandular stomach carcinogen in rats. The esophageal and non-grandular stomach fractions did metabolize N-nitrosopyrrolidine (NPYR) to a small extent, and liver microsomes and postmicrosomal supernatant metabolized both nitrosamines to a similar extent. Therefore, we advise caution in the interpretation of metabolic studies using 'target' and 'non-target' organs as indicative of activation of compounds to proximate carcinogens.

The half-times of the alpha-hydroxylated intermediates formed during metabolism of diethylnitrosamine and N-nitrosopyrrolidine have been determined. The method for determining half-times involved in vitro enzymatic conversion of the nitrosamine to a hydroxylated intermediate followed by trapping of the alkylating species generated from the chemical decomposition of the intermediate as it flowed through a column containing bound nucleophile. Half-times of less than 1 min at pH 7.4, 37 degrees C, were calculated from the distribution of alkylation on the column and the known flow rate of metabolic intermediates through the column. The half-times may be short enough to significantly limit organ distribution.

Twenty-one dietary and related chemicals have been evaluated for their potential inhibitory activities against the tumorigenic effects of N-nitrosopyrrolidine and N'-nitrosonornicotine using in-vitro metabolic assays in the target tissues, namely, rat liver microsomes and cultured rat oesophagus, respectively. Compounds studied include phenols, cinnamic acids, coumarins, isothiocyanates and indoles. Isothiocyanates were the most potent inhibitors of both nitrosamines in the acute studies, but were less active in chronic studies. This difference may be explained by the pharmacokinetic properties of these compounds. Phenols, cinnamic acids, coumarins and indoles were primarily inducers of N-nitrosopyrrolidine metabolism. The results suggest that isothiocyanates, in general, are the most promising chemicals for future study as protective agents against the carcinogenic effects of these nitrosamines.

The cytotoxic and mutagenic effects of increasing concentrations of N-nitrosopyrrolidine (NPYR) were studied using various DNA repair mutants of Escherichia coli together with rat-liver S9 activation system. Irrespective of which strain was used, the cytotoxic effects of NPYR were similar to those observed in the parent strain. Mutagenicity studies revealed that the uvrA- derivative was more mutable than its repair proficient parent. These observations suggest that NPYR reacts with DNA to generate bulky lesions, which although potentially mutagenic, do not contribute significantly to cell-killing. Subsequent experiments with the metabolic inhibitor SKF 525A revealed that this compound only partially inhibited the mutagenic activity of NPYR, suggesting that although hepatic mixed function oxidase enzymes may participate in NPYR activation other pathways of metabolism are also involved.

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.

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.

The metabolism of N-nitrosopyrrolidine (NPyrr) via alpha-hydroxylation is modified by pretreatments of the animals with compounds which affect the microsomal level of cytochrome P-450 and by addition, in vitro, of 2-diethylaminoethyl-2,2-diphenyl valerate hydrochloride (SKF 525-A), an inhibitor of cytochrome P-450. This phenomenon is due exclusively to the induction or the inhibition of the enzymatic activity involved in the microsomal metabolism. After preincubation in liquid medium, the mutagenic activity of NPyrr towards the Salmonella typhimurium strain TA 1530 is similarly modified by these effectors. A similar effect is not observed when using the plate incorporation method. The mutagenic intermediate is formed by the microsomal fraction. The presence of the S. typhimurium strain TA 1530 decrease the transformation of NPyrr into its ultimate metabolite (1,4-butanediol); there is a relationship between the formation of 1,4-butanediol and the mutagenic activity of NPyrr. The S. typhimurium strain TA 1530 is able to partially transform 4-hydroxybutanal, the first identifiable microsomal metabolite of NPyrr, into its ultimate metabolite (1,4-butanediol).

The effect of chronic ethanol consumption on the ability of isolated liver fractions to metabolize the carcinogen N-nitrosopyrrolidine (NPY) was examined. Microsomal fractions of treated animals exhibited increased rates of alpha-hydroxylation of NPY. Similar increases in the specific activities of aniline hydroxylase, reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase, and the specific content of cytochrome P-450 were also observed. In contrast, no differences in the specific activities of benzo(a)pyrene hydroxylase or glucose-6-phosphatase were observed. Liver postmitochondrial supernatants from ethanol-consuming animals were able to produce 5 times more mutants than did control preparations. It is concluded that alpha-hydroxylation of NPY is probably the mechanism by which NPY is converted to a mutagen and that this pathway can be induced by ethanol.

Cultured human bladder epithelial cells of normal origin are capable of metabolizing N-nitrosamines. Cultures from three different cell lines were incubated with NPYR and NEMA in order to investigate the formation of volatile N-nitrosamines. Seven volatile compounds with positive TEA response, including NDMA and NEMA, were detected in the culture media incubated with NPYR. The identity of NEMA, which was the main component of this group of metabolites, was confirmed by mass spectrometry. Culture media incubated with NEMA showed formation of NDMA. In four of five incubation experiments with NPYR, NHPYR was detected as a metabolite. The formation of metabolites containing the N-nitroso group presumably occurs via a beta-hydroxylation pathway.

The effect of modifiers of liver mixed function oxidase activity on the in vitro alpha-hydroxylation and mutagenicity of N-nitrosopyrrolidine (NPYR) has been examined in rats and hamsters. Hamster post-mitochondrial supernatants were able to convert NPYR to a mutagen more efficiently than rat preparations under all conditions studied. Aroclor 1254 pretreatment caused the greatest increase in mutagenic activity in both species while phenobarbital and 3-methylcholanthrene pretreatment gave intermediate values when compared to uninduced preparations. Microsomal alpha-hydroxylation of NPYR was induced by Aroclor 1254 pretreatment in both species. Pretreatment with 3-methylcholanthrene increased alpha-hydroxylation in hamsters but decreased it in rats. Phenobarbital pretreatment only slightly increased microsomal alpha-hydroxylation in either species. When microsomal alpha-hydroxylation rates were expressed per gram wet weight of liver, better agreement between rates of alpha-hydroxylation and mutagenicity in phenobarbital pretreated animals was obtained since inducer associated changes in total microsomal protein content were taken into account. An example of differential induction of an activation pathway (alpha-hydroxylation) and a degradative pathway (aldehyde dehydrogenase) is presented to illustrate a potential source of error in the interpretation of metabolic data obtained from post-microsomal supernatants and whole animal studies.

The mutagenicity of some N-nitrosodialkylamines, i.e. N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodi-n-butylamine, N-nitrosomorpholine and N-nitrosopyrrolidine, was assayed on Salmonella typhimurium TA100 by the pre-incubation method, and the effect of changing the pH of the pre-incubation mixture was examined. Markedly higher mutagenicities were observed when the pre-incubation of bacteria with nitrosamine and S9 mix was done at pH 5.2, compared with mutagenicities observable after the pre-incubations at conventional pH 7. Pre-incubations at pH 6.2 resulted in responses of intermediate strength. With phenobarbital-induced rat S9, the ratios of mutagenic potency found by the pH 5.2 pre-incubation to that found by the pH 7.2 preincubation were 15-30 for N-nitrosodimethylamine, 5-10 for N-nitrosodiethylamine, 10-20 for N-nitrosodi-n-butylamine, 2-3 for N-nitrosomorpholine and 4-6 for N-nitrosopyrrolidine. The mutagenic potency of each nitrosamine varied with the change of S9 source. The S9 sources examined were PCB-induced rat and mouse livers, and uninduced rat and mouse livers. No exceptions were observed for these S9 preparations regarding the higher mutagenicity at pH 5 than at pH 7. It is speculated that the higher mutagenicity observed by the pH 5 pre-incubation was due to the stability of the active intermediate, alpha-hydroxynitrosodialkylamines, in weakly acidic media.

The effect of chronic ethanol consumption by Syrian golden hamsters (SGH) on the carcinogenicity and target tissue metabolism of N-nitroso-pyrrolidine (NPYR) and N'-nitrosonornicotine (NNN) has been examined. Ethanol-consuming hamsters treated with NPYR developed more nasal cavity and tracheal tumours than controls. Ethanol consumption did not affect the carcino-genicity of NNN. When the metabolism of NPYR and NNN by isolated tracheal rings was examined, it was observed that tracheal rings isolated from ethanol-consuming SGH metabolized NPYR at a higher rate than similar preparations from control animals. In contrast, no differences in the rates of metabolism of NNN were observed.

When nursing Sprague-Dawley rats were treated with [14C]N-nitrosodimethylamine [(NDMA) CAS: 62-75-9], N-nitrosopyrrolidine (CAS:930-55-2), or N'-nitrosonornicotine (CAS: 16543-55-8), the liver and kidney DNA from their 14-day-old offspring that had been nursed over a 24-hour period became labeled. Upon analysis, liver DNA from sucklings whose nursing mothers were treated with [14C]NDMA showed N7-methylguanine- and O6-methylguanine-altered bases. The results suggest that these nitrosamines, which are present in food, tobacco smoke, and in different environmental sources, are a risk not only for lactating mothers but also for the nursing infants.

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.

Pretreatments with pyrazole, ethanol, nialamide or diethyldithiocarbamate were found to strongly depress the exhalation of 14CO2 and the incorporation of radioactivity in the acid-insoluble fraction of the liver in mice injected with N-[14C]nitrosopyrrolidine. Whole-body autoradiography performed with hemisections of mice at -80 degrees C (to prevent evaporation of the volatile N-nitrosopyrrolidine) and with dry tape-sections (to localize the non-volatile metabolites), using pretreated and non-pretreated mice, indicated a uniform distribution of the non-metabolized N-nitrosopyrrolidine in the tissues. At the shortest survival intervals (1 and 5 min), a high level of metabolites were found in the liver, the tracheo-bronchial and nasal mucosa and Harder's gland, indicating a local formation of metabolits in these tissues. At later survival intervals (0.5--24 h) metabolites were in addition found in tissues with a rapid cell turnover and a high rate of protein synthesis and in brown fat, which probably reflects incorporation of metabolites via normal biosynthetic pathways. Autoradiography of N-[14C]nitrosopyrrolidine in mice given the substance orally resulted in distribution pictures similar to those obtained after i.v. injections.