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Potent carcinogenicity of nitrosodiethanolamine in rats
Abstract
Nitrosodiethanolamine is found in synthetic cutting oils and in many cosmetic preparations and is probably the N-nitroso compound to which human exposure is greatest. It is formed by reaction of the commonly used amines diethanolamine and triethanolamine with nitrosating agents. An assessment of the possible risk in human exposure to nitrosodiethanolamine must be based on sound chronic toxicity data. A previously published chronic test of this compound in rats has shown it to induce liver tumours after very high oral doses, and tumours of the nasal cavity after administration of high repeated doses to Syrian hamsters by subcutaneous injection. To improve our understanding of the carcinogenic potency of nitrosodiethanolamine, we undertook a more extensive study, in which the compound was administered at concentrations ranging from 3,900 to 31,250 parts per million (p.p.m.) in drinking water, to groups of rats for about 6 months. We report here that when the animals were killed, all bore hepatocellular carcinomas, many of which metastasized at the higher doses, indicating that nitrosodiethanolamine is a carcinogen of considerable potency in the rat. However, it is inactive or very weakly active in short-term tests, such as the Salmonella mutagenesis test developed by Ames.
... Factory workers in the metalworking industry are exposed to cutting oils contaminated with A-nitrosodiethanolamine (Lijinsky et al., 1980). Experimental evidence has shown that nitrosamines are formed in vivo after the ingestion of harmless precursors (Ohshima and Bartsch, 1981) in a reaction influenced by the presence of activators and inhibitors of nitrosation. ...
Oesophageal cancer is an invariably fatal form of cancer with about 7000 deaths per annum in the UK alone. Evidence from the epidemiology and the mutation spectrum in the p53 gene suggests that the more common form of this cancer, squamous cell carcinoma, is caused by exposure to carcinogens. The N-nitrosamines are candidate carcinogens for this cancer in man. N-nitrosamines are metabolically activated by cytochromes P450 (P450s) and their organotropism is largely dependent on the distribution in the body of the particular nitrosamine and the P450s capable of metabolising it. The rat oesophagus is particularly susceptible to methylation and tumour formation by asymmetric N-nitrosamines, many of which selectively induce oesophageal tumours. This suggests that the oesophagus may contain a P450 that is absent or rare in other organs. The nature of this nitrosamine-metabolising P450 was investigated. A combination of Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Rapid Amplification of Cohesive Ends-Polymerase Chain Reaction (RACE-PCR) and oesophageal cDNA library screening showed the expression in the rat oesophagus of a novel P450 of the 2B subfamily. The deduced amino acid sequence of this P450 shows 84%, 83% and 77% identity to those of CYP2B1, CYP2B2 and CYP2B12 respectively. The catalytic activity of this new P450 is not yet known but metabolism and methylation studies in vivo using rats treated with phenobarbital, a CYP2B1/2B2 inducer, confirmed that these members of the 2B subfamily can metabolise the oesophagus selective nitrosamine, N-nitrosomethyl-n-butylamine, and that a major part of this metabolism was activating hydroxylation of the a-carbon of the butyl group. This supports the view that the novel P450 identified in rat oesophagus may be responsible for the metabolic activation and carcinogenicity of nitrosamines in the oesophagus. Further in vivo metabolic studies of N-nitrosomethyl-n-butylamine using untreated rats showed that it is metabolised in rat liver by CYP2E1. However, in this case 75% of the metabolism is detoxifying. An increase in methylation of oesophageal DNA by the nitrosamine after ethanol administration showed that CYP2E1 is not involved in the oesophageal metabolism of this nitrosamine.
... Diethylnitrosamine @EN) is a well documented hepatocarcinogen (Heath, 1962;Lijinsky et al., 1980). It is metabolized by hepatic mixed fimction oxidases, specifically P450 IIE 1, to an alkyl carbonium ion, which not only allcyIates DNA bases but also causes local oxidative stress (Swann and Magee, 1971). ...
... In 1977, it was reported that Nnitrosodiethanolamine (NDELA) had been found in cutting fluids (11), tobacco (12), cosmetics (13), lotions (13), and shampoos (13). Subsequently, NDELA was shown to be a carcinogen in the rat (14). Since these reports, scientists in industry and government have been working to develop and improve upon analytical methods for detecting NDELA (15). ...
... In 1977, it was reported that Nnitrosodiethanolamine (NDELA) had been found in cutting fluids (12), tobacco (13), cosmetics (14), lotions (14), and shampoos (14). Subsequently, NDELA was shown to be a carcinogen in the rat (15). Since these reports, scientists in industry and government have been working to develop and improve upon analytical methods for detecting NDELA (16). ...
... N-Nitrosodiethanolamine (see N -DEA structure) , is a potent liver carcinogen in experimental animals and is one of the most prevalent and abundant environmental occurring nitrosamines (Preussmann, 1982;Lijinsky, 1980). ...
(1) S-Nitrosothiole werden in vivo gebildet und sind an der NO-Signalübertragung beteiligt. In der vorliegenden Arbeit wurde in vitro die Schwefel-Stickstoff Transnitrosierung von S-Nitrosocystein (CysNO), S-Nitrosoglutathion (GSNO), S-Nitrosohomocystein (HCysNO), S-Nitrosocysteinylglycin (CGNO) und S-Nitroso-N-Acetylcystein (NACysNO) in der Reaktion mit dem sekundären Amin Diethanolamin (DEA) untersucht. Das resultierende N-Nitrosodiethanolamin (N-DEA), ein starkes Karzinogen, wurde ausgehend von CysNO und CGNO in Ausbeuten bis zu 11% gebildet. Hingegen war die Transnitrosierungsaktivität der anderen S-Nitroso-Verbindungen schwach ausgeprägt. Jedoch beschleunigte die Zugabe von L-Cystein zu einer Lösung von HCysNO und DEA den Abbau von HCysNO und führte zu einer signifikanten Bildung von N-DEA, die durch die gleichzeitige Entstehung von CysNO begleitet wurde. Somit können reaktive Nitrosothiole aus weniger reaktiven Analogen durch eine Schwefel-Schwefel-Transnitrosierung gebildet werden. Es wird vermutet, dass eine derartige Interaktion in vivo die NO-Signalübertragung beeinflusst. Diese Reaktion bietet einen alternativen Mechanismus zur Bildung von karzinogenen N-Nitrosoderivaten. NO· wird in den Zellen durch NO-Synthasen (NOS) gebildet. NO· und S-Nitrosothiole können möglicherweise mit O2 und O2·- zu Oxidantien wie N2O3 und ONOO.- reagieren. Diese modifiziern eine Vielzahl von Proteinen.
(2) mikrosomale Glutathion S-transferase (mGST) ist ein Mitglied innerhalb der Familie membranegebundener, multifunktionaler Proteine, welche an der zellulären Entgiftung von Xenobiotica und reaktiven Verbindungen endogenen Ursprungs beteiligt sind. Die mGST wurde hinsichtlich ihrer Aktivität sowie Aktivierung/Inhibierung durch N-Ethylmaleimid (NEM), CysNO oder Peroxynitrit charakterisiert. In der vorliegenden Arbeit wurde der Einfluß des S-Nitrosothiols, CysNO und Peroxynitrit auf die Aktivität von mikrosomaler Schweinnieren- und cytosolischer Rattenleber GST untersucht. NEM verursachte eine 5-fache Aktivierung der Rattenleber- bzw. 14%ige Inhibierung der Schweinnieren- mGST. Inkubation der Schweinnieren- mGST mit CysNO führte zu einer 9%igen Inhibierung, während die Rattenleber- mGST eine 2-fache Aktivierung zeigte. Exposition beider mGST-Formen mit Peroxynitrit resultiert in einer 23%igen Inhibierung bzw. in einer 2,2-fachen Aktivierung. Bemerkenswert ist, dass in Nieren mGST keine Stimulierung mit N-ethylmaleimid gesehen werden konnte. Schweinnieren- mGST und cytosolische GST der Rattenleber zeigten unterschiedliche Empfindlichkeiten gegenüber ONOO- und S-Nitrosothiolen.
(3) Der Einfluss des Homocysteins (HCys) auf die Stabilität von CysNO wurde im Serum- und in Phosphat-Puffer studiert. Ohne HCys wurde CysNO im Serum mit einer Halbwertszeit von weniger als 2,5 Minuten vollstän dig zerlegt, während die Halbwertszeit der S-Nitrosothiole auf 5 Minuten erhöht wurde, nachdem verschiedene Konzentration des Homocysteins der Mischung hinzugefügt wurden. Wenn CysNO in 10 mM Phosphat-Puffer bei pH 7 und 37°C inkubiert wurde, zerfielt es mit einer Halbwertszeit von 10 Minuten, wohingegen die Halbwertszeit des S-Nitrosothiols in der Gegenwart verschiedener Konzentrationen von Homocystein auf 80 min anstiegt. Daher erhöht der Transfer von NO-gruppen von S-Nitrosothiolen auf freie Thiole wie HCys die Stabilität von NO. Die überschüssige Zugabe von HCys zu CysNO fördert die Bildung von HcysNO sowohl in humanen Serum als auch in Pufferlösung. Dies kann wichtige physiologische und pharmakologische Bedeutungen haben.
(1) S-Nitrosothiols are formed in vivo and are involved in NO· signalling. In the present thesis the sulfur-to-nitrogen transnitrosation activity of S-nitrosocysteine (CysNO), S-nitrosoglutathione (GSNO), S-nitrosohomocysteine (HCysNO), S-nitrosocysteinylglycine (CGNO) and S-nitroso-N-acetylcysteine (NACysNO) in their reaction with the secondary amine diethanolamine (DEA) was investigated in vitro. The resulting N-nitrosodiethanolamine (N-DEA), a strong carcinogen, was formed in yields of up to 11% from CysNO and CGNO, whereas the transnitrosation activity of the other S-nitroso compounds was weak. However, the addition of L-cysteine to a solution of HCysNO and DEA accelerated the decomposition of HCysNO and led to a significant formation of N-DEA accompanied by the intermediate generation of CysNO. Thus, reactive nitrosothiols can be formed from less reactive analogs via sulfur-to-sulfur transnitrosation. It may be suggested that such interaction can affect NO· trafficking in vivo. The reaction provides an alternative mechanism for a generation of carcinogenic N-nitroso derivatives. NO· is produced in cells by nitric oxide synthases (NOS). NO· and S-nitrosothiols may react with O2 and O2·- to yield oxidants such as N2O3 and ONOO-. These oxidants may modify variety of proteins. (2) Microsomal glutathione S-transferase (mGST) is the membrane-bound member of the family of multifunctional proteins, involved in the cellular detoxification of xenobiotics and reactive endogenous compounds. The mGST has been characterized with regard to its activity profiles as well as activation/inhibition by N-ethylmaleimide (NEM), CysNO, or peroxynitite. In the present study, the influence of the S-nitrosothiol; CysNO and peroxynitrite on pig kidney microsomal and rat liver cytosolic GST activity was investigated. NEM caused 5-fold activation and 14% inhibition, of rat liver and pig kidney microsomal mGST, respectively. Incubation of pig kidney mGST with CysNO led to 9% inhibition, while the rat liver mGST showed 2-fold activation. Exposure of the pig kidney and rat liver mGST to peroxynitrite resulted in 23% inhibition and 2.2-fold activation, respectively. It is remarkable that in kidney mGST no stimulation with N-ethylmaleimide could be seen. Pig kidney mGST and rat liver cytosolic glutathione S-transferase showed different sensitivities towards ONOO- and S-nitrosothiols. (3) The influence of homocysteine (HCys) on the stability of CysNO in serum and phosphate buffer was studied. Without HCys, CysNO had decomposed completely in serum with a half-life of less than 2.5 min, whereas the half-life of S-nitrosothiols was increased to 5 min, when various concentration of homocysteine was added to mixture. When CysNO was incubated in 10 mM phosphate buffer, pH 7.0 at 37°C, CysNO decomposed with a half-life of 10 min, whereas the half-life of S-nitrosothiols was increased to 80 min in the presence of various concentration of homocysteine.
Therefore, the transfer of NO group from S-nitrosothiols to free thiols such as HCys strongly promoted the stability of NO. The excess addition of HCys to CysNO favours the formation HCysNO in both human serum and buffer. This may have important physiological and pharmacological implications.
... However, it is also relevant to compare the extent of metabolism of NMHA with that of the very poorly metabolized (34,35) NDELA since NMHA possesses both methyl and 2-hydroxyethyl groups. Although NDELA is also a potent hepatocarcinogen (36), it was reported not to be metabolized by rat microsomal enzymes (37). Therefore, alternative enzyme systems involving /3-oxidation or conjugation, such as those discussed above, have been speculated to be responsible for the carcinogenicity of NDELA (29,35,38). ...
The single-dose toxicokinetics of N-nitrosomethyl(2-hydroxyethyl)amine (NMHA) has been characterized in 8-week-old Fischer 344 rats by analysis using high-performance liquid chromatography of serial blood samples. An i.v. bolus dose of 0.6 mumol/kg to male rats revealed biphasic first-order elimination with a terminal half-life of 37.4 +/- 1.7 min for unchanged NMHA and 101 +/- 6 min for total radioactivity, and extensive conversion to polar metabolites was seen in the high-performance liquid chromatographic assays. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NMHA were 13.1 +/- 0.9 ml/min/kg, and 685 +/- 31 ml/kg, respectively. Renal blood clearance and intrinsic hepatic clearance were estimated to be 0.805 +/- 0.024 and 16.7 +/- 2.1 ml/min/kg, respectively. A similar dose given to female rats yielded a terminal half-life for NMHA of 27.2 +/- 1.2 min, a steady-state volume of distribution of 652 +/- 23 ml/kg, and systemic blood, renal blood, and intrinsic hepatic clearances of 16.9 +/- 1.3, 1.45 +/- 0.14, and 22.5 +/- 0.3 ml/min/kg, respectively. The sex differences in terminal half-life and systemic blood, renal blood, and intrinsic hepatic clearances were significant at the P less than 0.05 level. Larger doses given by gavage, which appeared to be completely absorbed from the gut, indicated systemic bioavailabilities for unchanged NMHA of 78 +/- 10% and 69 +/- 1% for male and female rats, respectively. Binding of NMHA to plasma proteins was found to be negligible. Taken together the data allow for the conclusion that the observed sex differences in toxicokinetic parameters are due to differences in the intrinsic hepatic clearance of the compound. This difference in the ability of the liver to metabolize NMHA in vivo correlates with and may contribute to the greater susceptibility of female rats to hepatocarcinogenesis and of male rats to development of tumors in the nasal epithelium following oral exposure to NMHA.
... In addition to being one of the A'-nitroso contaminants in certain pesticides (5), it is also found in tobacco products and tobacco smoke (6). Its carcinogenicity is well documented, first described by Druckrey et al. (7) and confirmed later by other authors (8)(9)(10). Since only relatively high doses of NDELA were used in the early carcinogenicity tests, Preussmann et al. (11) performed a dose-response study using five different dose levels including low exposure. ...
The carcinogenic efficiency of very low doses of N-nitroso-diethanolamine (NDELA), an N-nitroso compound of environmental significance, was assessed by administering it to male Sprague—Dawley rats at five different
dose levels: 0.2, 0.63, 1.5, 6 and 25 mg/kg b.w./day in the drinking water. Quantitation of the numbers and size of liver
foci positive for glucose-6-phosphate dehydrogenase (G6PDH) by morpho-metric methods revealed a good correlation between the
dose and duration of carcinogen treatment and the extent of G6PDH-positive foci development. Thus the area density increased
proportional to time and dose. The dose—time relation for the induction of 1% G6PDH-positive liver tissues assessed as a double
logarithmic plot gives a straight line with the same characteristics as that which results when the induction of liver tumors
is evaluated.
Human is continuously exposed to DBPs, and there is currently a large body of evidence on the health effects of DBPs in addition to the health risks from epidemiological studies. This chapter summarizes the toxic effects of DBPs in terms of in vivo, in vitro, and alternative toxicity tests. The in vivo toxicity tests detailed the carcinogenicity, reproductive and developmental toxicity, neurotoxicity, hepatotoxicity, and nephrotoxicity of DBPs, the in vitro toxicity tests reported cytotoxicity and its currently identified mechanisms, as well as genotoxicity, and the alternative toxicity tests described the toxic effects of DBPs in terms of three models, the zebrafish, the nematode, and the QSAR model. This chapter provides a comprehensive, in-depth, and systematic analysis and evaluation of the toxic effects of DBPs, which provides new approaches and insights for drinking water safety.
The nitrosamines belong to the class of N-nitroso compounds which also includes nitrosoalkylureas and nitroso-N-alkylcarbamates, conveniently described as nitrosamides, and nitrosamidines. The chemical structures of some examples of these compounds are shown in Figure 1. N-nitroso compounds undergo photochemical decomposition when exposed to ultraviolet light but the nitrosamines are chemically stable under physiological conditions in the absence of light. The nitrosamides and nitrosamidines, on the other hand, decompose rapidly at alkaline pH and, in some cases, in the presence of sulfhydryl compounds, to yield alkylating products. Decomposition may also occur more slowly at neutrality. These differences in chemical stability have profound effects on the biological activities of the compounds. More than 300 N-nitroso compounds are known to be carcinogenic and their chemical properties and biological activities, which include cytotoxicity, carcinogenicity, mutagenicity, teratogenicity and use in the chemotherapy of human cancer, have been extensively reviewed (1–7). Most studies of the acute toxicity of the nitrosamines have been carried out with the shorter chain dialkyl and simpler cyclic compounds such as dimethyl-and diethylnitrosamine and N-nitrosomorpholine which are selectively hepatotoxic while the nitrosamides and nitrosamidines cause tissue and cellular injury at the site of application and, in varying degree, to organs with rapid cell turnover such as the bone marrow, lymphoid tissue and intestine. As will be discussed later, these differences in tissue and organ specificity may reflect the requirement of the nitrosamines for metabolic activation and the absence of this requirement by the nitrosamides. More than 300 N-nitroso compounds have been found to be carcinogenic (4) and, so far all species adequately tested have proven to be susceptible to the carcinogenic action of one or more nitrosamines (8,9), N-nitrosodiethylamine having been the compound used most often. The susceptible species have included monkeys, dogs, cats, pigs, and the usual laboratory rodents, fish, amphibia, and most recently, snakes (9). The compounds are equally effective as mutagens in all of the usual test systems, with the nitrosamines requiring an enzyme system, usually the so-called liver S-9 mix of the Ames test, whereas the nitrosamides and nitrosamidines are direct acting mutagens (5–7). Several of the smaller molecular weight compounds are alkylating agents, the nitrosamines requiring metabolic activation and the nitrosamides acting directly to alkylate DNA and other cellular constituents.
Due to the development in the last decade of rapid and sensitive bacterial genetic test systems for detecting potential mutagens and carcinogens(1–3) there has been a recrudescence of testing of environmental chemicals for genotoxicity. Experiments performed in particular with theSalmonella/mammalian microsome test,(4,5) in which the ability of chemicals to cause reversion in auxotrophicSalmonella strains is determined on selective agar medium under the influence of various mammalian organ fractions, have yielded results on a variety of environmental compounds that show definite mutagenic activity in this assay system and are therefore to be considered as potentially mutagenic and carcinogenic in animals (see also Refs. 6 and 7).
Hepatocellular carcinomas are usually visible grossly and range in size from several millimeters to several centimeters (Farber 1976; Greenblatt and Lijinsky 1972; Goodman et al. 1994; Fig. 34). They are roughly spherical, although multilo-bulation may distort the shape (Fig. 35), and appear as single or multiple, light-tan to dark-red lesions in the liver. Rats chronically exposed to potent hepatocarcinogens frequently have multiple hepatocellular carcinomas alone or in combination with hepatocellular adenomas (Fig. 34). The liver of such an animal may be almost totally replaced by numerous, variably sized neoplastic and preneoplastic lesions.
Until this century, cancer was a rare disease, and there was no great interest in its nature or origin. Physicians were properly most concerned with the infectious diseases which carried off large proportions of the population, particularly the very young. Concern with those infectious diseases which kill a large portion of the population before middle age is still overwhelming in most of the world, especially in Asia and Latin America. With a few exceptions, cancer is a disease of older people and, therefore, a major problem only in the industrialized countries of Europe and North America. As survival rates improve in any country, it is likely that the incidence of cancer will increase and the urge to prevent this disease will become stronger, since cure is usually difficult and frequently impossible.
By use of GLC-ECD and HPLC-TEA techniques for N-nitroso compounds, N-nitroso-diethanolamine (NDELA) has been found in concentrations of 1.4–6.0 μg/m3 and 1.3–5.0μg/m3 respectively in all four air samples collected in the environment of a metalworking plant during metallurgical operations. NDELA was quantitated in air samples by GLC-ECD after converting it to its trifluoroacetyl derivative by reaction with the appropriate anhydride. NDELA was analyzed without derivatization in air samples using HPLC-TEA method. N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were also identified and later determined in two out of four air samples in concentrations of 0.08μg/m3 (for NDMA in both samples) and 0.14–0.16μg/m3 (for NDEA) using GLC-TEA procedure. The described method did not cause artifactual formation of N-nitrosomethyl-N-butylamine (NMBA) when methyl-N-butylamine was used as an internal marker of nitrosation during collection of NDELA in impinger traps.
The deamination of DNA bases by three alpha-nitrosaminoaldehydes, butylethanalnitrosamine, methylethanalnitrosamine, and N-nitroso-2-hydroxymorpholine (NHMOR), the direct metabolite of potent animal carcinogen N-nitrosodiethanolamine, was demonstrated by a set of in vitro experiments. The deamination of guanine, adenine, and cytosine bases in nucleotides, oligonucleotides, and calf thymus DNA gave xanthine, hypoxanthine, and uracil, respectively. The order of relative reactivities of the bases was as listed above. Deamination of cytosine to uracil was detected by the reaction of P-32-labeled oligonucleotide ([5'-P-32]CGAT) followed by enzymatic hydrolysis. Quantitative analysis of deamination of guanine and adenine in calf thymus DNA was performed by a gas chromatography/mass spectrometry-selected ion monitoring method. Both the extent and the rate of the deamination reactions which occur by transnitrosation from the alpha-nitrosaminoaldehyde to the base were determined for formation of xanthine and hypoxanthine. The deamination of guanine by NHMOR remained significant at low substrate levels.
The difference in the pK-values and chemical structure of N-nitrosodiethanolamine and triethanolamine in connection with a clear UV-maximum at 235 nm make possible a direct determination of N-nitrosodiethanolamine in triethanolamine up to 10 ng/ml possible when using an eluent of high optical transparency and a separating column with a high selectivity and reduced internal diameter. Linearity, reproducibility and accuracy meet the requirements to be fulfilled by trace-analytical methods.
The genetic activities of the carcinogen N-nitrosodiethanolamine (NDELA) were assayed somatically and germinally on two stocks carrying unstable alleles of the white (w+) eye colour gene: one incorporating a TE (z TE w+; coded UZ), the other with a tandem duplication of part of the encoding w+ sequence (wi16). Treatment was applied topically on late embryonic and early larval stages over a wide dose range (0.01–2.0 M) and the general mutagenic levels actually achieved were measured in terms of the X-recessive mutations (lethals and visibles) recovered in Muller-5 tests on samples of the males scored for somatic sectoring. The carcinogen was germinally ineffective on the z TE w+ and wi16 loci even at the maximal tested dose (2.0 M), and was only weakly mutagenic with respect tothe X-recessives (lethals + visibles) at massive doses (1.0–2.0 M). In contrast, it proved to be genetically effective somatically, inducing red eye sectors through regulatory effects involving the TE in the UZ stock and complete reversion to w+ in the case of wi16 at comparatively moderate doses (≤0.26 M). The possible implications of the demonstration of the genetic activity of NDELA in the soma to the carcinogenic process, and to the wider problem of screening for environmental genotoxic compounds, are discussed.
It was found that N-nitrosodiethanolamine (NDELA) was formed when diethanolamine (DELA) was heated in the presence of some inorganic pigments. Reactions were carried out as follows: a mixture of 30% (w/w) aqueous solution of DELA (1 mL) and pigment (0.3 g) was heated at 90 °C for 1 h. Carbon black, activated carbon, ferric ferrocyanide, and yellow iron oxide were shown to nitrosate DELA to form 180, 160, 35, and 1.0 μg of NDELA/1 g of pigment. NDELA was determined by high-performance liquid chromatography-thermal energy analysis (HPLC-TEA). The formation of NDELA was studied as a function of time, temperature, and pH. As to the effect of pH on the NDELA formation, different results were obtained between the case of some pigments and that of nitrite. In the gas desorbed from pigments with heating at 300 °C, nitrogen oxides (NOx) were detected and determined by the continuous Saltzman procedure. It was observed that NDELA was present after the reaction of NOx gas with DELA. On the basis of the data obtained, it was elucidated that NOx adsorbed on the pigments played the major role in the NDELA formation.
Two deuterated derivatives of N-nitrosodiethanolamine and two deuterated derivatives of N-Nitroso-2-hydroxymorpholine were prepared. 1,1,1′,1′-2H4-N-Nitrosodiethanolamine 1a was prepared in 99% isotopic purity by simple base catalyzed H-D exchange. 2,2,2′,2′-2H4-N-Nitrosodiethanolamine 1b was synthesized in 98% isotopic purity by the LiAlD4 reduction of dimethyl iminodiethanoate, followed by acid catalyzed nitrosation. 5,5-2H2-N-Nitroso-2-hydroxymorpholine 2a was prepared in 99% isotopic purity through a series of steps involving 1) the selective base catalyzed H-D exchange of the CH2 adjacent to the ester function of 2,2-diethoxyethylcarboethoxymethylnitrosamine, 2) its reduction with diisobutylaluminum hydride at −8°C followed by 3) acid catalyzed hydrolysis of the acetal and cyclization to the hemiacetal. 2-2H-N-Nitroso-2-hydroxymorpholine 2b (98% isotopic purity) was prepared through the LiAlD4 reduction of 2-hydroxyethylcorboethoxymethylnitrosamine at −78°C.
The impact of amines and/or potential nitrosating agents (nitrite and nitrate) present in water was studied in the amphibian Pleurodeles waltl (urodele). The genotoxic effects of secondary amines, atrazine (AT) and diethanolamine (DEIA), alone or in combination with sodium nitrite or sodium nitrate, were evaluated on peripheral erythrocytes using the newt micronucleus test.At the maximum concentration (MC) which could be tested, neither AT (0.3 ppm) nor DEIA (75 ppm) were found to be clastogenic. Likewise, even at their respective MC, neither sodium nitrite (140 ppm) nor sodium nitrate (8000 ppm) induced the formation of micronuclei. Negative results were systematically obtained under all conditions examined (pH 8, 6 or 5; alternation or indirect natural daylight and darkness or continuous darkness; prior incubation of both precursors in the dark for 48 h).The genotoxic effects of N-nitrosamines suspected to be formed from such precursors were also evaluated. Larvae were reared in water containing non-toxic concentrations of N-nitrosoatrazine (NAT) (3.75, 7.5 and 15 ppm) or N-nitrosodiethanolamine (NDEIA) (6.25, 12.5, 25 and 50 ppm). We found a significant difference in level of micronucleated erythrocytes between the lowest (3.75 ppm) and highest (15 ppm) concentrations of NAT. With NDEIA, a dose-response relationship was observed at concentrations from 12.5 to 50 ppm.We suggest that at the concentrations used (close to those which may be encountered in a polluted natural aquatic environment), if NAT or NDEIA are formed, the amounts produced are probably too low to yield a positive response in the newt micronucleus test.
S-Nitrosothiols are formed in vivo and are involved in NO signaling. We investigated the sulfur-to-nitrogen transnitrosation activity of S-nitrosocysteine, S-nitrosoglutathione, S-nitrosohomocysteine, S-nitrosocysteinylglycine and S-nitroso-N-acetylcysteine in their reaction with the secondary amine diethanolamine in vitro. The resulting N-nitrosodiethanolamine, a strong carcinogen, was formed in yields of up to 11% from S-nitrosocysteine and S-nitrosocysteinylglycine, whereas the transnitrosation activity of the other S-nitroso compounds was weak. However, the addition of l-cysteine to a solution of S-nitrosohomocysteine and diethanolamine accelerated the decomposition of S-nitrosohomocysteine and resulted in a significant formation of N-nitrosodiethanolamine accompanied by the intermediate generation of S-nitrosocysteine. Thus, reactive nitrosothiols can be formed from less reactive analogs via sulfur-to-sulfur transnitrosation. We suggest that this affects regulation of NO trafficking in vivo. The reaction provides an alternative mechanism for the generation of carcinogenic N-nitroso derivatives.
Epidemiologic evidence on the relationship between mineral oil exposure and cancer is reviewed. The review is restricted to occupations involving substantial dermal and inhalational exposure and for which an epidemiologic literature exists: metal machining, print press operating, and cotton and jute spinning. Mineral oils are complex mixtures of aliphatic hydrocarbons, naphthenics, and aromatics, the relative distribution of which depends on the source of the oil and the method of refinement. End-use products contain a variety of additives, and contamination by other agents generally occurs during use. Suspect agents include polycyclic aromatic hydrocarbons (PAH) (particularly benz[a]pyrene), nitrosamines, chlorinated paraffins, long-chain aliphatics, sulfur, N-phenyl-2-naphthylamine, and formaldehyde. The heterogeneity of this exposure makes epidemiologic study difficult and meta-analysis inappropriate. Nonetheless, several associations emerge from the literature with varying degrees of support. There is clear evidence that early formulations of mineral oils used in cotton and jute spinning and in metal machining were carcinogenic to the skin. Associations of mineral oil exposure with laryngeal and rectal cancer have received some support in the literature, particularly with respect to straight oils. Evidence is suggestive that grinding operations (which can entail either mineral oil-based or ethanolamine-based fluids) are associated with excess risk of cancer of the esophagus, stomach, and pancreas. A number of bladder cancer case-control studies have noted an association with work as a machinist. There is limited evidence of an association with cancer of the colon, prostate, and sinonasal region. Several studies of printers have yielded positive findings for lung cancer, whereas studies in metal machinists have been generally negative. The PAH and nitrosamine content of current formulations is lower than in the past and the implications of these changes in composition to the carcinogenicity of the formulations are not yet known.
Excesses of digestive and respiratory cancers have been reported previously in association with exposure to machining fluids, agents in widespread use as coolants and lubricants in machining operations. Previous studies have had limited power to distinguish the effects of the different types of machining fluids in use. In a cohort of over 30,000 workers employed at two automotive plants in Michigan, mortality patterns were studied in relation to exposure to each of the three major fluid types--straight oils, soluble oils, and synthetic fluids. Standardized mortality ratios were estimated for subgroups of the cohort ever exposed to each of the three fluid types, and Poisson regression analyses were used to assess trends in risk with duration of exposure. The data suggest modest positive associations between exposure to straight oils and rectal, laryngeal, and prostatic cancer and a negative association between soluble and synthetic fluid exposure and lung cancer.
Initiating activity of N-nitrosodiethanolamine (NDELA) for rat liver carcinogenesis was investigated using an 8-weeks bioassay system. Male F344 rats were initially treated with a single intraperitoneal injection of NDELA at one of five dose levels: 1,600, 800, 400, 200, or 100 mg/kg. Two weeks later, the rats were placed on 0.02% 2-acetylaminofluorene (2-AAF) or 0.05% phenobarbital (PB) containing diet for 6 weeks. All animals were subjected to 2/3 partial hepatectomy 4 weeks after the NDELA treatment, and killed at the end of the eighth week. NDELA itself exerted low toxicity in terms of body weight gain. Clear dose-dependent initiating activity of NDELA was observed in terms of development of glutathione S-transferase placental form (GST-P) positive liver cell foci, this being more apparent with PB promotion than with 2-AAF where the enhancing regimen itself caused multiple lesion development. Initiating potential of NDELA, however, was much lower than that observed for diethylnitrosamine in our previous work.
The potent carcinogen N-nitrosodiethanolamine (NDELA) was discovered as a contaminant of commercial metal-working lubricants over a decade ago. To determine whether or not improvements in industrial practice suggested in the meantime have eliminated this contamination from United States products, a selection of cutting fluids obtained from the current marketplace was analysed for NDELA content. All six semi-synthetic fluids examined contained NDELA at levels ranging from 0.5 to 4.3 ppm. Three of six petroleum-based lubricants and five of six synthetics also contained significant NDELA (when analysed at a detection limit of 0.03 ppm), at levels of up to 0.16 and 55 ppm, respectively. The mean concentrations were 1.5 ppm for the semi-synthetics, 0.07 ppm for the petroleum-based products, and 11.4 ppm for the synthetic metal-working fluids. While these levels are far below the values of 1-2% by weight (10,000-20,000 ppm) found in some contaminated products 13 years ago, they may nevertheless pose a continuing health risk for the machinists who work with them.
The effect of alcohol dehydrogenase (ADH/NAD) from yeast and horse liver was tested on the induction of chromosomal mutations and sister chromatid exchanges (SCE) by N-nitrosodiethanolamine (NDELA) in human lymphocyte cultures. BrdUrd (27 micrograms) was added 24 h after starting the cultures to allow visualisation of SCE. ADH/NAD and NDELA were added 24 h later in different concentrations. No significantly higher level of numerical or structural chromosome aberrations was observed. However, the SCE frequency per cell was significantly increased by adding NAD (31.25 mumol and 62.5 mumol). The exclusive addition of 220 units ADH from yeast as well as 1.8 units ADH from horse liver also raised the number of SCE highly significantly. The combination of NAD and ADH was more effective than each substances alone in the yeast but not in the horse liver system. NDELA in a range of 12.5-62.5 mumol, given to cultures with ADH/NAD from yeast, additionally increased the SCE frequencies in a dose-dependent way. Similar results were found in cultures containing ADH/NAD from horse liver and 6.25-31.25 mumol NDELA, but the total numbers of SCE were distinctly higher. These results indicate that NDELA is strongly activated by ADH from yeast but even more by ADH from horse liver.
A homemade rust-proof cutting fluid (RPCF) used in China was tested for carcinogenicity by an in vivo chronic experiment and for mutagenicity by the Ames Salmonella microsomal assay. Undiluted and threefold water-diluted fluid were given as drinking water to groups of young adult Wistar rats for 2 years. The treatment induced 11/40 malignant tumors with 9/40 acinar adenocarcinomas of the pancreas in the high-dose group. Simultaneous administration of ascorbic acid dissolved in the undiluted fluid at 2 g acid per 1 g sodium nitrite resulted in 1/40 pancreatic carcinoma. The results of the Ames test showed that the technical RPCF was mutagenic to TA100 with or without metabolic activation. It was concluded that the homemade RPCF, which is comprised of sodium nitrite, triethanolamine, and polyethylene glycol, may form direct-acting mutagen(s) upon storage and form, in vivo, e.g., nitrosamines that caused acinar pancreatic carcinoma in Wistar rats. Simultaneous administration of ascorbic acid is suggested for the protection of workers exposed to the rust-proof cutting fluid.
The mutagenicity of N-nitrosodiethanolamine (NDELA) and NDELA monoacetate was tested in vitro on lymphocytes of two healthy probands by determining the frequencies of chromosome aberrations, micronuclei and sister chromatid exchanges (SCE). A dose-dependent increase was found in all three test systems for NDELA as well as its monoacetate. The SCE test proved to be most sensitive for the genotoxic effect of NDELA because the differences to the control cultures had already become significant at 250-625 mumol/culture (26.6-65.4 mM). However, NDELA monoacetate showed a higher reactivity in the micronuclei and chromosome aberration test: significantly increased values were found even at 12.5 mumol (1.3 mM), whereas in the SCE test the differences became significant at the 25-mumol (2.7 mM) level. NDELA caused significantly increased rates of micronuclei and chromosome aberrations only at the highest test levels (625-1250 mumol; 65.4-127.6 mM). The results indicate important differences in the genotoxic effects of the two compounds, which might be explained by different lipophilicity and/or special activation processes.
For a large number of N-nitroso compounds a comparison of their carcinogenic effects in rats and Syrian golden hamsters has been made. Nitrosamines, which require metabolic activation, and nitrosoalkylamides, which do not, produce quite different tumor responses. There are also large differences in the types of tumor induced in rats and in hamsters. In all the studies doses of the various compounds, equimolar to the extent that was possible, are administered orally. Continuous doses (in drinking water or food) often produce a response different from that after administration of the same compound in pulsed doses (by gavage), even though the same total dose is delivered. Continuous doses of nitrosamines are usually more effective than pulsed doses, but with the nitrosoalkylureas, the reverse is more generally the case. Rat and hamster liver is a common target of many nitrosamines, but rarely of nitrosamides. The most common site of tumor induction in rats by N-nitroso compounds is the esophagus, but the hamster esophagus never responds. The pancreas duct of the hamster is a common target of nitrosamines containing a beta-oxygenated propyl group, but pancreas duct tumors are never seen in rats. Nitrosomethyl-n-alkylamines (with an even numbered carbon chain) induce bladder tumors in rats and hamsters. Many nitrosoalkylureas induce tumors of the nervous system in rats, as well as a great variety of other tumors. In hamsters, nitrosoalkylureas give rise only to tumors of the forestomach and spleen, but no tumors of the nervous system. The similar carcinogenic actions of certain groups of N-nitroso compounds can be related to their generation, directly or by metabolism, of similar simple moieties having certain organs as their target.
The in vivo alkylation of DNA by N-nitrosomethyl-(2-hydroxyethyl)amine (NMHEA) was examined in male and female F-344/N rats. NMHEA is a strong hepatocarcinogen in female rats when administered by gavage but a weaker hepatocarcinogen in male rats. Groups of 5 rats of each sex were treated by gavage with various doses of NMHEA dissolved in corn oil. After 4 h the animals were sacrificed and the livers, lungs, and kidneys were removed. The DNA from each liver was isolated and the neutral thermal and mild acid hydrolysates were separated by high-performance liquid chromatography. The alkylated guanines were quantified by fluorescence spectroscopy. NMHEA gives rise to four fluorescent alkylated guanines, 7- and O6-methylguanines, and 7- and O6-hydroxyethylguanines. The dose-response data revealed that all four lesions increased with dose. There was approximately 10x more methylation than hydroxyethylation at the 7 position of guanine. There was less O6 alkylation, but both methylation and hydroxyethylation were observed at all of the doses studied. The overall alkylation was the same in males and females at the 10- and 20-mg/kg doses, but at higher doses the females exhibited significantly higher levels of alkylation than males. The level of alkylation of DNA isolated from non-target tissues, lung, and kidney was low. The persistence of these lesions in vivo was studied at a dose of 25 mg/kg. Groups of five animals each were sacrificed at various times from 0 to 96 h. There was no significant difference between the sexes in persistence of any of the lesions in the liver. The 7-alkylguanines disappeared slowly over the observation period. 7-Methylguanine was present at 30% of the maximum level after 96 h, while 7-hydroxyethylguanine appeared to be more stable. The O6-alkylguanines were removed rapidly from the liver, being at base level by 48 h. The rapid removal of O6-hydroxyethylguanine suggests a repair process independent of O6-alkylguanine-DNA guanine alkyl transferase: an excision repair is postulated. In vitro alkylation of calf thymus DNA by N-nitrosomethyl-(2-tosyloxyethyl)amine, a surrogate for the putative O-sulfate conjugate of NMHEA, resulted in exclusive methylation of DNA-guanine at both the 7 and O6 positions; no hydroxyethylation was detected. In vitro alkylation of calf thymus DNA with 2-hydroxyethyl-ethylnitrosourea resulted in exclusive hydroxyethylation of DNA-guanine at the 7 and O6 positions.(ABSTRACT TRUNCATED AT 400 WORDS)
The effects of lifetime treatment with dimethylaminoethanol on longevity and cryptogenic neoplasm formation were studied in females of two mouse sub-lines, the C3H/HeN which carries a germinal mammary tumor provirus and the C3H/HeJ(+) which also carries the exogenous mammary tumor virus. Administration in the drinking water of 10 mM dimethylaminoethanol to the C3H/HeN mice or 15 mM to the C3H/HeJ(+) mice did not result in significant differences between treated and untreated groups in average survival. No changes in age-related organ structure or morphology were observed with dimethylaminoethanol treatment, except for an apparent decrease in the amount of lipofuscin in the liver judged in histological sections. Among untreated C3H/HeJ(+) females, 89% developed neoplasms of the mammary gland, ovary, liver, lung and reticuloendothelial system, while the incidence was 88% in the treated mice. In C3H/HeN females, neoplasms of the mammary gland, ovary, liver, lung and lymphatic system occurred in 57% and in 60% of treated mice. Also, there was no statistically significant difference between control and treated animals in the age of onset or the type of specific neoplasms. Dimethylaminoethanol did not induce any neoplasms.
A comparison has been made of the carcinogenic effects of nitroso-2,6-dimethylmorpholine and several hydroxylated acyclic nitrosodialkylamines derived from it or related to it in rats and Syrian hamsters. In rats nitrosodimethylmorpholine was the most potent, inducing mainly esophageal tumors. Nitrosodiethanolamine was the weakest of the five nitrosamines in both rats and hamsters. Tumors of the pancreas ducts were induced by four of the five compounds, but only in hamsters, and esophageal tumors appeared only in rats. Most of the nitrosamines induced tumors of liver and lung in both rats and hamsters. A study of alkylation of nucleic acids of the liver following treatment of rats and hamsters with the radiolabeled nitrosamines showed that nitrosodiethanolamine alkylated liver nucleic acids in rats to only a very small extent. The other four nitrosamines all gave rise to 7-methylation and O6-methylation of guanine residues in DNA of hamster liver and all but nitrosodimethylmorpholine in rat liver DNA, which corresponded quite well with the induction of liver tumors in the two species. Quantitatively, however, there was not a good correlation between liver DNA alkylation and the potency of the nitrosamine in inducing tumors.
The effect of the mixed-function oxidase inhibitor phenylimidazole (PI) and the amine oxidase inhibitors iproniazid (IPRO) and aminoacetonitrile (AAN) on the mutagenic activity of various carcinogens was determined in intrasanguineous host-mediated assays, using mice as hosts and E. coli 343/113 as an indicator of mutagenic activity. The carcinogenic compounds dimethyl-, diethyl-, methylethyl-, and diethanolnitrosamine (DMNA, DENA, MENA, and DELNA respectively) and 1,2-dimethylhydrazine (SDMH) were administered i.p. to mice pretreated or not with one of the inhibitors. After 4 h exposure to each of the carcinogens, E. coli cells recovered from the liver of non-pretreated mice showed considerable induction of VALr mutations; after pretreatment of the hosts with the three inhibitors, significant reduction of the amounts of induced mutants in vivo was observed. Particularly, PI proved a very efficient inhibitor of DENA, MENA, DELNA, and SDMH mutagenicity (93%-97% reduction), suggesting that these carcinogens are mainly activated by cytochrome P-450-dependent enzymes. However, since PI might also inhibit the NAD-mediated activation of DELNA by alcohol dehydrogenase (ADH), the present experiments do not rule out an additional role of ADH in the in vivo mutagenic activation of DELNA. AAN and IPRO were less and much less effective, respectively, in reducing the mutagenic activity of all compounds. Surprisingly, PI showed less inhibition of the mutagenic activity of DMNA (60% reduction), as compared to the other carcinogens; this indicates that metabolic routes other than the cytochrome P-450-dependent enzyme system may be important for the activation of DMNA.
The mutagenic activity of diethanolnitrosamine (NDELA), a carcinogenic compound which leads to inconsistent results in standard in vitro procedures was tested in vitro and in animal-mediated assays with the indicator strain Escherichia coli (E. coli) K-12 343/113. This strain allows the simultaneous detection of forward and back mutations arising in several genes of the E. coli chromosome. In animal-mediated assays in which mice were used as hosts for i.v. injected E. coli indicator cells, s.c. application of NDELA induced a dose dependent increase of galactose fermenting mutants in cells recovered from galactose fermenting mutants in cells recovered from the livers of animals exposed for 3 h to the mutagen.
Comparison with results obtained with diethylnitrosamine (DENA) in the same test system revealed that the two compounds apparently cause different types of mutagenic lesions. Induction of arg+ mutations by DENA and several other aliphatic nitrosamines is mainly due to base pair substitutions, whereas NDELA is rather mutagenic in the galRs system. This latter system is, in addition, sensitive to frameshifts and deletions. These differences in mutagenic specifity suggest that NDELA and DENA, although structurally closely related, are activated via different molecular mechanisms.
In fact, evidence is accumulating that alcohol dehydrogenase (ADH) could be involved in the activation of NDELA. On the other hand, the effective mutagenesis of NDELA obtained in vitro with E. coli upon addition of rat liver microsomal fraction would not be expected if ADH is involved in the activation since the S-9 Mix used in the present experiments was devoid of cofactors (NAD, NADP), necessary to accomplish oxidation by ADH.
Therefore, further in vivo studies were performed, in which pyrazole, a potent blocker of ADH, was administered prior (1 and 24 h) to the injection of the mutagen. The observation that a dose dependent increase of mutants in the liver (and to a lower extent in the spleens) of treated animals takes place under conditions in which ADH activity is blocked, whereas several microsomal enzymes are stimulated, indicated that besides oxidation of NDELA by ADH other metabolic activation pathways are involved.
Apparently enzymes contained in the liver homogenate, possibly NADPH dependent enzymes of the microsomal ethanol oxidizing system, play an important role in the formation of mutagenic metabolites of NDELA.
The metabolism of N-nitrosodiethanolamine (NDELA) was studied to assess whether the formation of the beta-oxidated metabolites N-(2-hydroxyethyl)-N-(formylmethyl)nitrosamine (EFMN) and N-(2-hydroxyethyl)-N-(carboxymethyl)nitrosamine (ECMN) is involved in the mechanism of tumor induction in various animal species with different susceptibility to NDELA carcinogenicity. In vitro studies using liver S9 fractions from rats, hamster, B6C3F1 and CD-1 mice and rabbits showed that all the animal species metabolize NDELA through the beta-oxidation pathway, although to different extents. Urinary excretion of NDELA and its metabolite ECMN in rats, hamsters and mice after 5 mg X kg-1 NDELA i.p. confirmed these findings. The results suggest there is no correlation between carcinogenesis by NDELA and its beta-oxidation. The possibility that ECMN formation might represent a detoxifying metabolic pathway for NDELA is discussed.
The carcinogenic potential of triethanolamine was examined in F344 rats. Triethanolamine was dissolved in distilled water at levels of 0 (control), 1, and 2%, and groups of 50 males and 50 females were given these doses ad libitum as drinking water for 2 yr. The dose levels in females were reduced by half from wk 69, because of associated nephrotoxicity. A variety of tumors developed in all groups, including the control group, and all tumors observed were histologically similar to spontaneous tumors in this strain of rats. No statistically significant increase of the incidence of any tumor was observed in the treated groups of both sexes by the chi-square test. In this study, however, there was an increase in nephrotoxicity, which appeared to have an adverse effect on the life expectancy of the treated animals, especially of females. Therefore, an age-adjusted statistical analysis on incidences of main tumors or tumor groups of both sexes was also done by methods recommended by Peto et al. (1980). The result showed that a positive trend (p less than 0.05) was noted in the occurrence of hepatic tumors (neoplastic nodule/hepatocellular carcinoma) in males and of uterine endometrial sarcomas and renal-cell adenomas in females. These tumors, however, have been observed spontaneously in this strain of rats, and their incidences in the control group of the present study were lower than those of our historical controls. These results may indicate that a positive trend in the occurrence of these tumors is not attributable to triethanolamine administration. Increased incidence of renal tumors in the female high-dose group may have been connected with renal damage. Histological examination of renal damage observed in the treated groups, especially in the female high-dose group, revealed acceleration of so-called chronic nephropathy. In addition, mineralization of the renal papilla, nodular hyperplasia of the pelvic mucosa, and pyelonephritis with or without papillary necrosis were also observed. Thus, it is concluded that under these experimental conditions triethanolamine is not carcinogenic in F344 rats but is toxic to the kidneys.
Whole-body autoradiography in Sprague-Dawley rats injected i.v. with [14C]N-nitrosodiethanolamine ([14C]NDELA) showed a localization of tissue-bound radioactivity in the liver and the nasal olfactory mucosa. Microautoradiography of the nasal olfactory mucosa showed the highest labelling over the subepithelial glands (Bowman's glands) in the lamina propria mucosae. Experiments in vitro showed a capacity of the liver and the nasal mucosa to form 14CO2 from the [14C]NDELA. Most of the injected [14C]NDELA was recovered in the urine in a non-metabolized form. A small proportion of the dose was exhaled as 14CO2. The target tissues for the NDELA carcinogenesis in Sprague-Dawley rats are the liver and the nasal mucosa. Our results indicate that a bioactivation of the NDELA will take place in the nasal mucosa, as well as in the liver.
The genetic activities of the carcinogen N-nitrosodiethanolamine (NDELA) were assayed somatically and germinally on two stocks carrying unstable alleles of the white (w+) eye colour gene: one incorporating a TE (z TE w+; coded UZ), the other with a tandem duplication of part of the encoding w+ sequence (wi16). Treatment was applied topically on late embryonic and early larval stages over a wide dose range (0.01-2.0 M) and the general mutagenic levels actually achieved were measured in terms of the X-recessive mutations (lethals and visibles) recovered in Muller-5 tests on samples of the males scored for somatic sectoring. The carcinogen was germinally ineffective on the z TE w+ and wi16 loci even at the maximal tested dose (2.0 M), and was only weakly mutagenic with respect to the X-recessives (lethals + visibles) at massive doses (1.0-2.0 M). In contrast, it proved to be genetically effective somatically, inducing red eye sectors through regulatory effects involving the TE in the UZ stock and complete reversion to w+ in the case of wi16 at comparatively moderate doses (less than or equal to 0.26 M). The possible implications of the demonstration of the genetic activity of NDELA in the soma to the carcinogenic process, and to the wider problem of screening for environmental genotoxic compounds, are discussed.
The ability of N-nitrosodiethylamine (DENA) to induce transformation of the mammary cells was studied in culture of the whole
mammary organ from BALB/c female mice. Incidence of nodule-like alveolar lesions (NLAL) in the glands in vitro has been used as a measure of transformation. NLALs are analogous to the precancerous hyperplastic alveolar nodules (HAN)
of mouse mammary gland in vivo. The mammary glands were treated with graded concentrations (0.1–2.5 μg/ml) of DENA during lobuloalveolar morphogenesis in
medium (Waymouth's MB752/1) containing insulin, prolactin, hydrocortisone and aldosterone. DENA treatment caused a dose-related
increased occurrence of NLAL in the glands in vitro and concentration of 1.5 μg/ml produced the highest incidence of 85%. The high incidence of NLAL was accompanied by a 3-fold
increase of DNA repair activity in the DENA treated glands. Incubation of the glands for 6 days after DENA treatment in medium
containing N-(4-hydroxyphenyl)retinamide and the same hormone mixture caused 61% inhibition of NLAL incidence. The results
indicate that DENA is capable of inducing a high level of transformation of the mammary epithelial cells in vitro and that this retinoid can inhibit expression of the transformed cells acting at the promotional level.
PIP
Analysis of 7 patients 20-30 years of age treated for hepatic cell carcinoma during the past 15 years at Massachusetts General Hospital indicated that 5 had an androgenous or exogenous disturbance of gonadal function. The 2 men in this series were taking methyltestosterone. Of the women, 2 had been taking oral contraceptives (OCs) for long durations, 1 had Stein-Leventhal syndrome, and 1 was infertile for 18 months. 3 of the 4 women with altered reproductive function had fibrolamellar carcinomas. These cases suggest that abnormalties of hypophyseal-gonadal endocrine metabolism can predispose to the development of hepatic cell carcinoma. Cessation of methyltestosterone or OC use and major hepatectomy apparently cured 3 of these patients. Although the data are consistent with a role for sex steroids in some cases of hepatic carcinogenesis, a case-control study would be needed to eliminate chance occurrence of fibrolamellar-variant cancer in the age group most likely to be using OCs. It is likely that hepatic carcinogenesis is a complex process involving genetic susceptibility, drug potency, regenerative urges, and the cooperation of promoters and co-carcinogens.
N-Nitrosodiethanolamine (NDELA), a potent carcinogen, has not so far been found to be mutagenic in a wide range of test systems. In particular, mutagenicity testing in Salmonella typhimurium with rat liver S-9 mix or microsomal fraction used for activation has failed to indicate mutagenicity. However, when incubated with alcohol dehydrogenase (ADH) in the presence of NAD, NDELA is converted to a potent mutagen. A possible mechanism of activation comprises the generation of an aldehyde as a primary metabolite formed by NAD/ADH and its subsequent rearrangement into cyclic intermediates. The latter might either be further metabolized or spontaneously decompose into various alkylating agents and glycolaldehyde. Standard test conditions used for the Ames test will not favor the detection of mutagens to be activated by NAD/ADH because they require the presence of NADPH, whereas ADH needs NAD to become an activating enzyme, as shown for NDELA.
Hepatocytes were isolated from the livers of fed rats and incubated with a mixture of glucose (10 mM), ribose (1 mM), mannose (4 mM), glycerol (3 mM), acetate (1.25 mM), and ethanol (5 mM) with one substrate labelled with 14C in any given incubation. Incorporation of label into CO2, glucose, glycogen, lipid glycerol and fatty acids, acetate and C-1 of glucose was measured at 20 and 40 min after the start of the incubation. The data (about 48 measurements for each interval) were used in conjunction with a single-compartment model of the reactions of the gluconeogenic, glycolytic and pentose phosphate pathways and a simplified model of the relevant mitochondrial reactions. An improved method of computer analysis of the equations describing the flow of label through each carbon atom of each metabolite under steady-state conditions was used to compute values for the 34 independent flux parameters in this model. A good fit to the data was obtained, thereby permitting good estimates of most of the fluxes in the pathways under consideration. The data show that: net flux above the level of the triose phosphates is gluconeogenic; label in the hexose phosphates is fully equilibrated by the second 20 min interval; the triose phosphate isomerase step does not equilibrate label between the triose phosphates; substrate cycles are operating at the glucose-glucose 6-phosphate, fructose 6-phosphate-fructose 1,6-bisphosphate and phosphoenolpyruvate-pyruvate-oxaloacetate cycles; and, although net flux through the enzymes catalysing the non-oxidative steps of the pentose phosphate pathway is small, bidirectional fluxes are large.
Male CD-COBS rats were given N-nitrosodiethanolamine (NDELA) by intravenous or cutaneous administration at a dose of 5 mg/kg. Blood and liver were analysed for NDELA at various times after administration. The excretion of unchanged NDELA and its acidic metabolite N-(2-hydroxyethyl)-N-carboxymethylnitrosamine (ECMN) was determined in urine for 24 hr after treatment. The semilogarithmic blood concentration-time plot after iv injection showed a triphasic profile indicating that a three-compartment model may adequately describe the kinetics of this compound. After cutaneous application NDELA was rapidly absorbed through the skin, and the absolute bioavailability was calculated to be 27% from blood data, and to be 32% (NDELA + ECMN) from urine data. Hepatic NDELA levels reflected blood levels after both treatments, indicating that this organ does not accumulate NDELA to a significant extent. Urinary excretion of unchanged NDELA after iv and cutaneous administration was 83 and 25% of the administered dose, respectively. ECMN excretion was 4.9 and 2.5% of the administered dose after iv and cutaneous administration, respectively.
A dose-response study of the carcinogenicity of N-nitrosodiethanolamine was conducted in F344 rats. The nitrosamine was administered in drinking-water at a controlled rate (20 ml/rat/day, 5 days/wk) to groups of 16 or 20 rats of each sex. The doses administered were as follows: 2500 mg/litre drinking-water for 45-wk, 1000 mg/litre for 50 wk, 400 mg/litre for 50 wk and 400 mg/litre for 75 wk. Almost all of the treated animals died with hepatocellular carcinomas, of which 20-45% metastasized in each group. The females in each treatment group tended to die earlier, because they received a higher dose per unit body weight, but for each sex there was a dose-related decrease in survival as the carcinogen dose increased. There was a considerable incidence of tumours of the nasal cavity. The incidence of these tumours tended to be higher in males than in females and might be sex- or lifespan-related. A number of rats had cholangiocarcinomas in the liver and there were a few kidney tumours and tumours of the oesophagus in animals given the higher doses. None of these tumours was seen in the untreated controls of either sex, almost all of which outlived the treated animals. It is concluded that N-nitrosodiethanolamine is a carcinogen of considerable potency in rats.
In simulated metalworking coolants that contained both nitrite and di- or triethanolamine at pH 9, N-nitrosodiethanolamine formed at an initial rate of 11 or 6 ppm/wk, respectively. This rate was increased on heating the fluids, on acidification or by the addition of paraformaldehyde, 1,3,5-trimethylhexahydro-s-triazine, ferricyanide or ferric ethylenediaminetetraacetate. N-Nitrosodiethanolamine also formed when nitrite-free coolants containing either of the two amines above were exposed to nitric oxide in air. No nitrosamines were detected in fluids containing primary amines in place of the secondary and tertiary amines, except that N-nitrosooxazolidine was formed in the fluid containing monoethanolamine after addition of formaldehyde-releasing agents, and N-nitrosodiethanolamine and N-nitrosomorpholine were found in fluid containing diglycolamine (HOCH2CH2OCH2CH2NH2) after the fluid was heated at 100 degrees C for 48 hr. These data suggest several steps by which nitrosamine formation in commercial cutting fluids might be substantially reduced: avoiding acid-splitting as a disposal procedure; removing nitrite from the fluid and/or scavenging adventitious nitrosating agents; avoiding unnecessary heating; adding preservatives to the diluted fluid rather than to the commercial concentrate; replacing inherently nitrosatable amine additives by substitutes which are resistant to nitrosamine formation; minimizing concentrations of catalytically active metal complexes.
Nitrosodiallylamine has been reported to be non-carcinogenic in rats while nitrosodipropylamine and nitrosodiethanolamine are liver carcinogens. That nitrosodipropylamine is metabolized at the alpha-position by liver microsomes from Fischer-344 rats supports the widely held contention that such metabolism is responsible for the carcinogenicity of nitrosamines. Nitrosodiallylamine is also metabolized at the alpha-position by the same microsomal preparations. Thus, although alpha-oxidation may be responsible for the carcinogenicity of some nitrosamines, this mechanism alone cannot account for tumorigenicity. Nitrosodiethanolamine is not metabolized by rat liver microsomes, but is metabolized by hepatocytes for Fischer-344 rats. In this case, a mechanism other than the oxidation at the alpha-position may be responsible for the carcinogenic action.
25 aliphatic nitrosamines were examined in the Ames assay for bacterial mutagens, using rat liver "S-9" for activation. Of them, 8 carcinogens were mutagenic and 5 non-carcinogens were not mutagenic. However, 2 compounds not carcinogenic in rats were mutagenic and 9 carcinogens were not mutagenic, including 6 that are liver carcinogens in rats.
N-Nitrosodiethanolamine (NDEIA), a compound known to produce liver tumours in rats, was detected in widely used consumer products such as cosmetics, hand and body lotions and hair shampoos. The concentration varied from less than 1 ng/g (ppb) to 48,000 ng/g, the latter in a facial cosmetic. The source of the NDEIA was presumably the nitrosation of the di- and/or triethanolamine additives. NDEIA was identified by coincidence of retention time on three different high-pressure liquid Chromatograph columns using an N-nitrosamine-specific detector. In a single case the compound eluting at the retention time of NDEIA was also isolated and identified by high-resolution mass spectrometry.
N-Nitrosodiethanolamine (NDEA) and 1,1-diethanolhydrazine (DEH) were synthesized and injected subcutaneously weekly in male and female Syrian golden hamsters. The total NDEA dose per hamster was approx. 15 g/kg body wt. applied in either 7 or 27 subdoses. DEH was administered in 78 applications to two groups yielding total doses of 1.1 g and 273 mg/kg body wt. Under these conditions, DEH did not show a specific demonstrable carcinogenic effect. However, within 78 weeks after the first application, 39 out of 56 hamsters treated with NDEA developed tumors. Primarily, neoplasms of the nasal cavity and tracheal tumors were observed, as well as a few hepatocellular adenomas and sarcomas at the injection site. These findings and those of the earlier study on carcinogenicity of NDEA in rats raise concern as to the safety for human consumption or industrial use of products with the potential for forming NDEA.
N-nitrosodiethanolamine has been found to be present at a concentration of 0.02 to 3 percent in several brands of synthetic
cutting fluids. Its identity was confirmed by three independent techniques: (i) by measuring the retention times on two different
high-performance liquid-chromatography columns, (ii) by dehydration to N-nitrosomorpholine, and (iii) by preparation of the
O-methyl ether derivative.