Search

To test the hypothesis that N-nitrosodimethylamine (NDMA) and possibly other nitrosamines are synthesized naturally in the gastrointestinal tract, the urinary excretion of nitrosamines has been measured in people whose first pass clearance and metabolism of nitrosamines had been inhibited by administration of ethanol. Five fasted volunteers consumed 350 mg of nitrate in beet juice followed, 1 h later, by a volume of 10% alcohol in carbonated water sufficient to raise their blood alcohol concentrations to 50-80 mg/100 ml. This alcohol concentration was then maintained over a 6 h period. No NDMA or any other volatile nitrosamines were excreted in urine during this 6 h period or during the subsequent 12 h (detection limit = 0.01 mumol). These results suggest that less than 0.5 mumol NDMA, formed by endogenous nitrosation in the fasting human, is likely to be present in the stomach at any time.

Endogenous nitrosation of proline was investigated in smokers and nonsmokers. Volunteers consumed a volume of beet juice equivalent to 325 mg nitrate, and 1 hour later they consumed 500 mg proline. In separate experiments volunteers ingested proline alone. Twenty-four-hour urine samples were collected and analyzed for N-nitrosoproline. When proline alone was ingested, there was no significant difference in urinary nitrosoproline excretion between smokers and nonsmokers. When beet juice and proline were consumed, however, smokers produced approximately 2.5 times as much N-nitrosoproline as nonsmokers. Salivary thiocyanate levels were approximately 3.2 times higher in smokers compared to those in nonsmokers. Salivary nitrite levels of smokers and nonsmokers, either before or after beet juice consumption, were not different. Salivary nitrate concentrations, however, were higher in nonsmokers than in smokers after beet juice consumption but not before. Our results suggest that the higher level of salivary thiocyanate in smokers is responsible for the increased rate of endogenous nitrosation of proline in this group compared to the rate in nonsmokers. Nitrosating agents in cigarette smoke do not appear to play a significant role.

The rat hepatocarcinogen nitrosodimethylamine and the esophageal carcinogens nitrosomethylbenzylamine and nitrosomethylamylamine were shown to produce chromosomal damage, as manifested by micronucleus formation, in their target tissues. There was cross-reactivity in the two tissues, however, at high dose levels. Nitrosodiethylamine, which produces tumors in both the liver and esophagus in the rat, also produced micronuclei in both tissues.

Analysis of 24 h urine samples of rats administered N-nitrosodipropylamine (NDPA), N-nitroso-2-hydroxypropylpropylamine (NHPPA) or N-nitroso-2-oxopropylpropylamine (NOPPA) showed that the major excretion product in each case was the beta-glucuronide of NHPPA. This conjugate was shown to be present at levels of about 5%, 80% and 50% of the administered dose for NDPA, NHPPA and NOPPA, respectively, based on exhaustive hydrolysis of the urine samples with beta-glucuronidase. Generally only low levels (less than 2%) of the unchanged nitrosamines or their unconjugated metabolites (NHPPA for administered NDPA and NHPPA for administered NOPPA) were detected. We were unable to detect N-nitrosomethylpropylamine or N-nitrosodimethylamine in the urine of any of the animals.

The nitrosamines exhibit remarkable species and tissue specificity in their carcinogenic action (Shank and Magee, 1981). The biological basis of this specificity, however, is poorly understood. A primary target for many nitrosamines, particularly unsymmetrical dialkylnitrosamines, is the rat esophagus. These nitrosamines induce high incidences of basal and squamous cell tumours, independent of the route of administration.

The reduction of N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-oxopropyl)propylamine (NOPPA) by hepatic and pancreatic cytosol and microsomes from Syrian golden hamsters and Sprague-Dawley rats has been examined. All hepatic fractions reduced both substrates, although the activity depended on the fraction tested and the cofactor employed (NADH or NADPH). Generally, hamster hepatic fractions contained higher activity than the rat hepatic fractions and BOP was a better substrate than NOPPA. Of the pancreatic fractions, only cytosol exhibited reductase activity. The hamster cytosol was able to utilise both cofactors, but the rat fraction exhibited activity only when NADPH was present. BOP was the better substrate for the pancreatic enzymes and in the presence of NADPH, the rat and hamster activities were about equal. These results suggest that the pancreatic reduction of BOP to HPOP is unlikely to be a significant factor in the species-specific induction of pancreatic cancer by BOP.

The melatonin in urine samples from six healthy adult volunteers was concentrated on Amberlite XAD-2 resin, eluted with organic solvents, and quantitated by use of a bioassay technique (the dermal melanaphore response of larval anurans to melatonin in their bathing medium). The melatonin content of samples collected between 11 p.m. and 7 a.m. was, in each case, several times higher than that of samples collected between 7 a.m. and 3 p.m. or between 3 p.m. and 11 p.m.

Methylation in vitro of calf thymus DNA, a supercoiled plasmid, poly(dG) · poly(dC), and poly(dGdC) · poly(dGdC) by N -nitroso(acetoxymethyl)methylamine and N -nitroso(acetoxybenzyl)methylamine in the presence of esterase, and by N -nitrosomethylurea was investigated. Although there were differences in the amounts of 7-methylguanine and O 6 -methylguanine formed in the various DNA substrates, the methylation pattern was the same for each of these methylating agents. The three compounds reacted identically when methylation of a portion of a 345 bp restriction fragment of the plasmid pBR322 was examined at nucleotide resolution by a sequencing assay. They also showed a tendency to react preferentially with particular guanines. These data suggest that the three N -nitroso compounds methylate DNA via a common intermediate such as the methyl diazonium ion, which exhibits some sequence specificity.

Male Sprague-Dawley rats were pretreated with various chemicals to determine their effects on the microsomal activation of the esophageal carcinogen N-nitrosomethylbenzylamine [( NMBzA ) CAS: 937-40-6; N-methyl-N- nitrosobenzylamine ] in the rat esophagus and, for comparative purposes, in the rat liver. When rats were pretreated with NMBzA , little change in hepatic NMBzA - debenzylase activity was observed. In contrast, NMBzA metabolism in the esophagus was significantly (60-65%) reduced. Similarly, pretreatment of rats with disulfiram [CAS: 97-77-8; bis( diethylthiocarbamoyl )disulfide] caused a 40% decrease in esophageal metabolism, but it had no significant effect in the liver. Pretreatments with the methylenedioxybenzenes safrole [CAS: 94-59-7; 4-allyl-1,2-(methylenedioxy)benzene], isosafrole [CAS: 120-58-1; 1,2-(methylenedioxy)-4-propenylbenzene], and dihydrosafrole (CAS: 94-58-6; 1,2-(methylenedioxy)-4- propylbenzene ) caused a marked induction (twofold to fivefold) of the hepatic metabolism of NMBzA , but again esophageal metabolism was suppressed. The results indicate that esophageal metabolism of NMBzA is either unchanged or suppressed by the various chemical pretreatments, but hepatic metabolism of the nitrosamine is induced by the methylenedioxybenzenes .

The immobilization of Rhodospirillum rubrum chromatophores was successfully performed by entrapping them in polyacrylamide. Their photophosphorylating activity was about 40% of native chromatophores. The temperature and pH optima for immobilized chromatophores were similar to the native photosynthetic apparatus and kinetic parameters showed that the rate of photophosphorylation in polyacrylamide particles was diffusion controlled. Light penetration of the gel particles was not a limiting parameter. Immobilization considerably increased the stability of the chromatophores towards denaturation.

Some chemically induced tumors in rodents are associated with single point mutations in ras genes1. It is important to know whether these mutations arise at sites in DNA of unusually high reactivity for ultimate carcinogens, or whether carcinogen interaction is random. This information is not available for simple methylating agents such as those derived from N-nitroso compounds.

The syn and anti conforms of N-nitrosoproline, N-nitrososarcosine and N-nitroso-2-(ethylamino)-ethanol, have been separated by liquid chromatography. These conformers result from hindered rotation about the NN bond. Separation was achieved using adsorption, reversed-phase, and ion-exchange modes. For the nitroso-amino acids, a shift in the equilibrium conformer concentration was observed with changes in pH.

Using an in vitro assay system for measuring 7-methylguanine formation in DNA, it was demonstrated that N-nitroso(2-oxopropyl)propylamine (NOPPA) is converted into a methylating agent by a microsomal, cytochrome P-450 dependent mixed function oxidase from rat liver. Formation of propionaldehyde, but not formaldehyde, in this assay system, indicated that a single alpha-oxidation reaction on the propyl side chain of NOPPA leads to the formation of a methylating agent. A mechanism for this reaction is proposed.

Publisher Summary This chapter discusses the separation of folic acid derivatives and pterins, by high-performance liquid chromatography. For this study, folic acid, 7,8-dihydrofolic acid, 5,6,7,8-tetrahydrofolic acid, 5-methyltetrahydrofolic acid, and p-aminobenzoylglutamic acid were purchased from Sigma Chemical Co. 5,10-Methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid were prepared by the method of Rabinowitz. The 5,10-methylenetetrahydrofolic acid was prepared by the method of Huennekens et al. N(10)‑nitrosofolic acid was prepared by the method of Cosulich and Smith. 2‑Hydroxy-N(10)-nitrosofolic acid and 2-hydroxyfolic acid were prepared by the method of Angier et al. Xanthopterin, pterin, and pterin-6-carboxylic acid were purchased from Sigma. Pterin-6-carboxaldehyde was prepared by the method of Waller et al. Because most reduced derivatives of folic acid are extremely labile to oxidation, by air, 2-mercaptoethanol is normally added to the solutions, containing 7,8-dihydro and 5,6,7,8-tetrahydro derivatives. 2-Mercaptoethanol absorbs at 254 nm and interferes with the detection of chromatographic peaks. This chapter discusses the separation of the three oxidation states of folic acid. Separation of N(5)-and N(10)-substituted tetrahydrofolates is described in the chapter. Separation of the products of nitrosation of folic acid is also elaborated in the chapter in detail.

Administration of N-nitrosodi-n-propylamine to rats leads to the formation of 7-n-propylguanine but not 7-isopropylguanine in hepatic DNA. For RNA, a small amount of the rearranged adduct is formed. Alkylation of DNA and RNA therefore appears to occur primarily via a bimolecular reaction rather than a unimolecular pathway involving free alkyl cations.

Metabolism of N-nitrosodi-n-propylamine by an isolated rat liver microsomal fraction yielded 17% isopropanol and 83% n-propanol (expressed as a percentage of total propanol formed). Base-catalyzed decomposition of N-n-propyl-N-nitrosourea yielded 39% isopropanol and 61% n-propanol. The values provide evidence for involvement of carbocations in both of these reactions.

Pancreatic islets of Langerhans of the Syrian hamster were maintained in culture for as long as 43 wk. Islets were prepared by collagenase/hyaluronidase digestion of minced pancreas. The islets quickly attached to the plastic culture flasks and lost their spherical form as they flattened out to form circular monolayers. Few fibroblastoid cells were observed. As outward migration continued, the islets became vacoulated with the ultimate formation of monolayer rings. Throughout the culture period the beta cells continued to synthesize and secrete insulin. Furthermore, the cells maintained a responsiveness to glucose stimulation with increased rates of hormone secretion in the presence of elevated concentrations of the sugar. These studies demonstrate the suitability of Syrian hamster islets for studies involving long-term culture.

We have demonstrated that ammonia is oxidized to nitrate in the rat. Male Sprague-Dawley rats gavaged with 1000 mumol N-15-ammonium chloride each day for 5 days were found to excrete low, but significant amounts of excess N-15-nitrate in their urines on the five days of treatment and on the five subsequent days. We recovered a total of 0.28 +/- 0.03 mumol excess N-15-nitrate (mean +/- SE) per rat, which indicates that ammonia is converted to nitrate in a yield of approximately 0.0080%. The oxidation of N-15-labeled glycine and L-glutamic acid to N-15-nitrate could not be detected. N-15-Hydroxylamine was oxidized in the rat to N-15-nitrate in a yield of 4.7%, which shows that hydroxylamine is a possible intermediate in the ammonia oxidation process. Injection of rats with Arochlor 1254, an inducer of several isozymes of cytochrome P-450, did not significantly affect the rate of endogeneous nitrate synthesis. Carbon tetrachloride, which causes hepatic lipid peroxidation, produced a small but significant increase in nitrate synthesis. We confirmed the observation that a bacterial endotoxin can greatly stimulate nitrate synthesis, and we showed that concurrent treatment with superoxide dismutase does not modify the effect of the endotoxin. An in vitro chemical model system was used to demonstrate that oxidation of ammonia to nitrate by the hydroxyl radical at physiological pH is chemically feasible. Our results are consistent with the hypothesis that ammonia is oxidized to nitrate in vivo by a non-enzymatic process which involves active oxygen species such as the hydroxyl radical. We estimate that a 215 g rat produces 3.0 mumol of nitrate per day via ammonia oxidation.

An inhibitor of Clostridium perfringens formed when low levels of nitrite were autoclaved with a defined chemical medium. A systematic study of the medium revealed that only amino acids and mineral salts were involved in the production of this inhibitor, which was proven to be a toxic compound formed from cysteine, ferrous sulfate, and sodium nitrite. The inhibitor was compared to several known compounds. S -nitrosocysteine inhibited the test organism, but would not form in the test system in amounts large enough to explain the observed inhibition. Roussin red salt was unstable in the test system and therefore was not the inhibitor. Roussin black salt, which was also inhibitory, could form in sufficient amounts to explain the inhibition. A complex of cysteine, iron, and nitrie oxide was detected in the autoclaved solution of cysteine, ferrous sulfate, and sodium nitrite; this cysteine complex did not appear to be inhibitory, however, at levels which could form in the autoclaved medium. The observed inhibition may have been due to the combined effects of sublethal concentrations of each compound.

alpha-Acetoxy-N-nitrosopyrrolidine (AcO-NPYR), in the presence of esterase, was reacted with the plasmid pBR322. The AcO-NPYR-treated plasmid was nicked by reaction with spermidine, suggesting the presence of apurinic sites. The treated plasmid also blocked the action of the restriction endonuclease BglI and, to a lesser extent, DraI. The recognition sequences of these restriction enzymes suggest that AcO-NPYR interacts preferentially with guanine and/or cytosine bases in DNA.

Reaction of diethylnitrosamine in the non-enzymatic, ascorbic acid-dependent, hydroxylating system of Udenfriend yielded N-nitroso-2-(ethylamino)-ethanol as the major product extracted into methylene chloride. The major product derived from nitrosopiperidine in the same system was N-nitroso-4-piperidone. These products, however, were mutagenic to Salmonella typhimurium TA 1535 only when activated by a rat liver microsomal preparation.

The tumor initiating activities of nitrosomethylbenzylamine (NMBzA), an esophageal carcinogen, and nitrosodimethylamine (NDMA), a hepatocarcinogen, were compared in rat liver using modifications of the initiation assays of Tsuda et al. (Cancer Res., 40, 1157, 1980) and Pitot et al. (Nature, 271, 456, 1978). Equimolar doses of NMBzA or NDMA (33.5 mu-mol/kg) were injected i.p. into male Sprague-Dawley rats 18 h after partial hepatectomy. Following selection, foci of preneoplastic hepatocytes were detected by histochemical staining for gamma-glutamyltranspeptidase. Nitrosomethylamylamine (NMAmA), 115 mumol/kg), also an esophageal carcinogen, was tested in the assay of Tsuda et al., and nitrosodiethylamine (NDEA, 33.5 mumol/kg), a hepatic and esophageal carcinogen, was tested in the assay of Pitot et al. Both NDMA and NDEA induced significant increases in the number of preneoplastic foci above background. In contrast, neither NMBzA nor NMAmA increased the number of foci above background. Microsomes from regenerating liver had a lower capacity to metabolize both NDMA and NMBzA compared to microsomes from intact liver, but the decrease in activity was similar for both compounds. Neither NDMA nor NMBzA significantly inhibited the first wave of DNA synthesis in vivo in the regenerating liver. The results demonstrate that in contrast to NDMA and NDEA, NMBzA and NMAmA lack tumor initiating activity in the liver.

Adenosine triphosphate (ATP) plays a prominent role in many biosynthetic pathways wherein chemical bonds are made which otherwise would not form in significant quantity in dilute aqueous solution. In such reactions the breakdown of ATP is coupled via a common intermediate with synthesis of the new chemical bond. The work reported here is part of a coordinated effort to demonstrate large-scale, cell-free enzymatic synthesis of useful products with simultaneous regeneration of the ATP consumed in the biosynthetic reaction.

Creatine reacts with nitrite under acid conditions to produce first sarcosine and then N-nitrososarcosine, which is a weak carcinogen in the rat. Creatinine reacts with acidified nitrite to produce either creatinine-5-oxime or 1-methylhydantoin-5-oxime, depending on reaction conditions. The toxicity and environmental significance of these compounds is not yet known.

Summary 1. Populations of introduced species are often thought to perform differently, or experience different population dynamics, in their introduced range compared to their native habitat. Differences between habitats in climate, competition or natural enemies may result in populations with varying density dependence and population dynamics. 2. We examined the long‐term population dynamics of the invasive common wasp, Vespula vulgaris, in its native range in England and its invaded range in New Zealand. We used 39 years of wasp density data from four sites in England, and 23 years of data from six sites in New Zealand. Wasp population time series was examined using partial rate correlation functions. Gompertz population models and multivariate autoregressive state‐space (MARSS) models were fitted, incorporating climatic variation. 3. Gompertz models successfully explained 59–66% of the variation in wasp abundance between years. Density dependence in wasp populations appeared to act similarly in both the native and invaded range, with wasp abundance in the previous year as the most important variable in predicting intrinsic rate of increase (r). No evidence of cyclic population dynamics was observed. 4. Both the Gompertz and MARSS models highlighted the role of weather conditions in each country as significant predictors of annual wasp abundance. The temporal evolution of wasp populations at all sites was best modelled jointly using a single latent dynamic factor for local trends, with the inclusion of a latent spring weather covariate. That same parsimonious multivariate model structure was optimal in both the native and invaded range. 5. Density dependence is overwhelmingly important in predicting wasp densities and ‘wasp years’ in both the native and invaded range. Spring weather conditions in both countries have a major influence, probably through their impact on wasp colony initiation and early development. The population dynamics in the native range and invaded range show no evidence of cyclic boom‐and‐bust dynamics. Invasive species may not exhibit different population dynamics despite considerable variation in abundances throughout their distribution.

In view of the known toxicological properties of nitrosamines with laboratory animals and the uncertainties concerning their danger toward humans, the authors feel the instructive value of this reaction probably does not warrant its continued use, especially in situations where special precautions cannot be employed.