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L K Keefer

Publications and source records attributed to L K Keefer.

At least 73 records · Page 4Linked to original sources
PubMed1991

Complexes of .NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects.

Selected nucleophile/nitric oxide adducts [compounds which contain the anionic moiety, XN(O-)N = O] were studied for their ability to release nitric oxide spontaneously in aqueous solution and for possible vasoactivity. The diversity of structures chosen included those in which the nucleophile residue, X, was that of a secondary amine [Et2N, as in [Et2NN(N = O)O]Na, 1], a primary amine [iPrHN, as in [iPrHNN(N = O)O]Na, 2], a polyamine, spermine [as in the zwitterion H2N(CH2)3NH2+(CH2)4N[N(N = O)O-](CH2)3NH2, 3], oxide [as in Na[ON(N = O)O]Na, 4], and sulfite [as in NH4[O3SN(N = O)O]NH4, 5]. The rate constants (k) for decomposition in pH 7.4 phosphate buffer at 37 degrees C, as measured by following loss of chromophore at 230-260 nm, were as follows: 1, 5.4 x 10(-3) s-1; 2, 5.1 x 10(-3) s-1; 3, 0.30 x 10(-3) s-1; 4, 5.0 x 10(-3) s-1; and 5, 1.7 x 10(-3) s-1. The corresponding extents of nitric oxide release (ENO) were 1.5, 0.73, 1.9, 0.54, and 0.001 mol/mol of starting material consumed, respectively, as determined from the integrated chemiluminescence response. Vasodilatory activities expressed as the concentrations required to induce 50% relaxation in norepinephrine-constricted aortic rings bathed in pH 7.4 buffer at 37 degrees C (EC50) were as follows: 1, 0.19 microM; 2, 0.45 microM; 3, 6.2 microM; 4, 0.59 microM; and 5, 62 microM. Vasorelaxant potency (expressed as 1/EC50) was strongly correlated with the quantity of .NO calculated from the physicochemical data to be released in the interval required to achieve maximum relaxation at the EC50 doses (r = 0.995). This suggests that such nucleophile/.NO adducts might generally be useful as vehicles for the nonenzymatic generation of nitric oxide, in predictable amounts and at predictable rates, for biological purposes. The particular significance for possible drug design is underscored in the very favorable potency comparison between several of these agents and the established nitrovasodilators sodium nitroprusside and glyceryl trinitrate (EC50 values of 2.0 and greater than 10 microM, respectively) in parallel aortic ring tests.

Animals↗
PubMed1991

Beta-deuteration of N-nitrosoethylmethylamine causes a shift in DNA methylation from rat liver to esophagus.

While N-nitrosoethylmethylamine (NEMA) is carcinogenic primarily for the liver, its beta-trideuterated derivative, N-nitroso( [2-D3]ethyl)methylamine (NEMA-d3), also produces a high incidence of tumors in the esophagus. To determine whether this shift in organ specificity is associated with an altered pattern of DNA alkylation, [methyl-14C]- and [1-ethyl-14C]-labeled NEMA-d3 were administered to adult male Fischer 344 rats as a single i.p. dose (0.05 mmol/kg; 4 h survival). Levels of methylated and ethylated purines in the DNA of various organs were determined by radio-chromatography on Sephasorb-HP columns. When compared to previous data using undeuterated NEMA, 7-methylguanine levels were found to be reduced by approximately 30% in liver and kidney, but were 160% greater in esophagus. This resulted in a decrease in the 7-methylguanine ratio for liver/esophagus from 109 to 29. O6-Methylguanine was diminished in liver and kidney, but levels in lung and esophagus were too low for quantitative detection. Similarly, deuteration led to an 18% decrease of 7-ethylguanine in hepatic DNA. The observed increase in esophageal DNA methylation correlates with the increased carcinogenicity of NEMA-d3 relative to undeuterated NEMA in that organ. Since pharmacokinetic studies have shown that beta-trideuteration of NEMA does not alter its bioavailability, the data suggest that the observed shift in target organ results from isotopically-induced changes in the balance among competing metabolic pathways in different rat tissues.

Animals↗
PubMed1991

Etiological research on gastric cancer and its precursor lesions in Shandong, China.

Research over the past several years in an area of Shandong, China, with one of the world's highest rates of gastric cancer, has yielded clues to the environmental determinants of this tumour. Interviews with 564 gastric cancer patients and 1131 population-based controls revealed increased risks associated with consumption of sour pancakes, a fermented staple unique to the area, in samples of which volatile N-nitrosamines have been detected. Lower risks were found among people who had a higher intake of fresh vegetables, including garlic and other Allium vegetables which contain constituents that can inhibit carcinogenesis by N-nitrosamines and other substances in experimental animals. A pilot study involving assays of urine and gastric juice from 60 individuals in a screening programme showed higher levels of N-nitrosoproline and of cis- and trans-N-nitroso-2-methylthiazolidine 4-carboxylic acid among persons with gastric dysplasia than in either normal controls or those with chronic atrophic gastritis. We are trying to characterize the transition and progression of precursor lesions to gastric cancer and to evaluate the role of dietary variables, nutrients, N-nitroso compounds and other factors in particular stages of the carcinogenic process.

China↗
PubMed1990

Metabolic denitrosation of N-nitrosodimethylamine in vivo in the rat.

Enzymatic denitrosation is a potentially inactivating metabolic route that has been shown to convert carcinogenic N-nitrosodimethylamine (NDMA) to methylamine (MA) in vitro. To investigate its quantitative course in vivo, groups of 8-week-old male Fischer rats have been given small (8-15 mumol/kg) p.o. or i.v. bolus doses of 14C-labeled NDMA and the subsequent formation of radioactive MA has been monitored by high performance liquid chromatographic analysis of serially collected blood samples from each individual. Adjusting the [14C]MA fluxes observed for the previously measured rates at which MA is itself eliminated from the system after intragastric administration, denitrosation was calculated to represent a rather uniform 21.3 +/- 1.3% (SE) of total NDMA elimination in the four animals studied. By contrast, repetition of the experiment with fully deuterated NDMA (NDMA-d6) revealed a significantly wider variance in the results (39.8 +/- 8.9%). An alternative calculation using values for elimination of i.v. doses of MA and its trideuteromethyl analogue gave an even larger difference for MA formation between NDMA and NDMA-d6, the estimated extents of in vivo denitrosation in this case being 14.5 +/- 0.9% and 48.3 +/- 10.8%, respectively. The results indicate that denitrosation is a major metabolic pathway for NDMA elimination and suggest that deuteration of the carcinogen induces a shift in its metabolism toward increasing denitrosation at the expense of the competing activation pathway. Consequently, denitrosation may be the previously undefined in vivo metabolic route, the existence of which was suggested by the findings that deuteration of NDMA lowered its hepatocarcinogenicity and liver DNA alkylating ability in rats.

Animals↗
PubMed1990

Single-dose toxicokinetics of N-nitrosomethylethylamine and N-nitrosomethyl (2,2,2-trideuterioethyl)amine in the rat.

To investigate the origins of an organotropic shift toward increasing esophageal carcinogenicity and DNA alkylation caused by beta-trideuteration of the hepatocarcinogen, N-nitrosomethylethylamine (NMEA), the single-dose toxicokinetics of NMEA and N-nitrosomethyl(2,2,2-trideuterioethyl)amine (NMEA-d3) has been characterized in 8-week-old male 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 rats revealed biphasic first order elimination with a terminal half-life of 9.46 +/- 0.69 min for unchanged NMEA and 28.9 +/- 2.4 min for total radioactivity. Extensive conversion to polar metabolites was observed in the chromatograms. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NMEA were 39.9 +/- 4.6 ml/min/kg and 496 +/- 36 ml/kg, respectively. There was negligible plasma protein binding and no detectable NMEA was excreted unchanged in the urine. Larger doses given by gavage indicated a systemic bioavailability of 25 +/- 1%. Similar doses of NMEA-d3 given to other groups of rats revealed no significant differences in any of the toxicokinetic parameters. No N-nitrosomethyl(2-hydroxyethyl)amine was found as a detectable metabolite of NMEA or NMEA-d3 in any of the blood or urine samples which were analyzed. When considered together, the data suggest that previously observed differences in organ specificity for the carcinogens, NMEA and NMEA-d3, are not due to differences in the total amounts of nitrosamine reaching particular tissues, but may have other localized causes such as differences in the enzymes responsible for metabolism which are present in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Persistence of N-nitrosodiethanolamine contamination in American metal-working lubricants.

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.

Diethylnitrosamine↗
PubMed1989

Sex differences in the single-dose toxicokinetics of N-nitrosomethyl(2-hydroxyethyl)amine in the rat.

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.

Animals↗
PubMed1988

Carcinogenicity of deuterium-labeled 1,2-dimethylhydrazine in mice.

To study the effect of deuterium substitution on the carcinogenicity of 1,2-dimethylhydrazine (DMH) in mice, two comparisons were made between DMH and its fully methyl-deuterated analogue, [2H6]DMH. In a lifetime study with the CBA strain, groups of 19-30 animals of each sex were dosed s.c. weekly with 8 mg/kg of either DMH or [2H6]DMH for 8, 16, or 32 weeks. In the second study, female CF-1 mice were given DMH or [2H6]DMH in 10 weekly s.c. doses of 12 mg/kg each (13.2 mg/kg for [2H6]DMH) and examined for colon tumors 36 weeks after the first dose. Deuteration significantly decreased tumor incidence in the colon of males (P less than 0.01) and the anal tissue of both sexes (P less than 0.05) but increased that of hepatomas and lung tumors in males (P less than 0.01). Substrate deuteration did not significantly affect overall incidence of any other tumor type, however, including hemangioendotheliomas and kidney tumors in both sexes, as well as colon, uterine, ovarian, liver, and lung tumors in females. The results indicate that C--H bond breakage is kinetically important in the activation of DMH to its ultimately carcinogenic form in organs such as the male colon (relative risk in DMH-versus [2H6]DMH-treated animals approximately equal to 6), and that inhibition of this process by substrate deuteration allows a diversionary mechanism having a smaller isotope effect to become relatively more extensive. The qualitatively different effect in other organs (e.g., kidney, relative risk approximately equal to 1) supports recent suggestions that the net mechanism of activation can differ from one target tissue to another, possibly by striking a different balance between parallel metabolic pathways. The lack of a significant isotope effect on overall colon tumor incidence in females of either strain suggests that differences in relative importance among competing enzymes may also be responsible for sexual dimorphism in tumor induction by DMH.

1,2-Dimethylhydrazine↗
PubMed1988

Decontamination and disposal of nitrosoureas and related N-nitroso compounds.

An improved procedure for chemically decontaminating residues of nitrosoureas and related N-nitroso compounds ("nitrosamides") commonly used in the cancer research laboratory is proposed. Treatment of accumulated wastes with aluminum:nickel alloy powder while progressively increasing the basicity of the medium consistently led to at least 99.98% destruction of each nitrosamide tested. Hazardous diazoalkanes were never detected in yields of greater than 0.1%. The mutagenicity of the completed reaction mixtures was never more than 3 times background except when the N-nitroso compound contained a 2-chloroethyl group. In most cases, the completeness of reaction could be determined chromatographically, not only to demonstrate the disappearance of the starting N-nitroso compound, but also to follow production of identifiable products in sufficient abundance to account for the starting material destroyed; none of the organic products observed was mutagenic in any of the four tester strains used. The procedure described herein proved reliable in two checker laboratories besides our own when applied to mixtures of seven N-nitroso compounds: N-methyl-N-nitroso-p-toluene-sulfonamide; N-methyl-N-nitrosourethane; N-methyl-N-nitrosourea; N-methyl-N'-nitro-N-nitrosoguanidine; N-ethyl-N-nitrosourea; N-ethyl-N'-nitro-N-nitrosoguanidine; and N-ethyl-N-nitrosourethane. All of the other procedures investigated for destruction of nitrosamides, including the widely used approach of dissolving the nitrosamides in alkali, were associated with important disadvantages.

Chemical Phenomena↗
PubMed1988

Carcinogenesis in rats by cyclic N-nitrosamines containing sulphur.

The effects of chronic exposure to three sulphur-containing heterocyclic N-nitrosamines were determined after repeated oral administration to female Fischer 344 rats. Nitrosothiazolidine did not significantly affect the survival of the rats or the incidence of tumours at a total dose of 3.5 mmol. Nitrosodithiazine, an analogue of nitrosothiazolidine which contains an extra sulphur atom inserted between the carbons of its CH2-CH2 moiety, produced only three tumours (two of the nasal mucosa) in a group of 20 rats at a total dose of 1.75 mmol/rat. Nitrosothialdine, the all-cis 2,4,6-trimethyl analogue of nitrosodithiazine, was a potent carcinogen that significantly shortened the lifespan and produced oesophageal tumours in 70% of treated rats as well as numerous tumours of the tongue and liver; this outcome was unexpected because alpha-methyl substitution in other heterocyclic nitrosamines usually reduces or eliminates tumorigenicity. The results extend the data base on the carcinogenic activity of molecules containing both divalent sulphur and the nitrosamino function. The lack of significant carcinogenicity of nitrosothiazolidine in this study suggests that its presence in the human food supply presents a relatively minor risk.

Animals↗
PubMed1987

Deuterium isotope effect on denitrosation and demethylation of N-nitrosodimethylamine by rat liver microsomes.

In an attempt to elucidate the molecular basis for the decrease in rat liver carcinogenicity and DNA-alkylating ability that accompanies deuteration of N-nitrosodimethylamine (NDMA), NDMA and its fully deuterated analogue ([2H6]NDMA) were incubated with acetone-induced rat liver microsomes. Rates for the competing metabolic routes, denitrosation and demethylation, were determined from colorimetric data on nitrite and formaldehyde generation, respectively. The Vmax calculated for demethylation of NDMA was 7.9 nmol/min/mg, while that for denitrosation was 0.83 nmol/min/mg. Deuteration of NDMA did not significantly change the Vmax for either pathway, but it did increase the Km for demethylation from 0.06 to 0.3 mM. The Km for denitrosation was also increased from 0.06 to 0.3 mM on deuteration, as determined by incubating an equimolar mixture of amino-15N-labeled NDMA with [2H6]NDMA and measuring the methyl[15N]amine:[2H3]methylamine ratio by derivatization-gas chromatography-mass spectrometry. The fact that the Km values for denitrosation were so similar to those for demethylation suggested that the two pathways were catalyzed by the same enzyme. The isotope effects calculated from these data [VmaxH/VmaxD approximately 1 and (Vmax/Km)H/(Vmax/Km)D approximately 5] show that microsomal metabolism of NDMA is not significantly shifted from demethylation to denitrosation on deuteration of substrate and may indicate a low commitment to catalysis for the enzyme. The results are consistent with the view that the metabolism of NDMA is initiated by formation of an alpha-nitrosamino radical which either combines with a hydroxyl radical to form the alpha-hydroxynitrosamine as the initial product of the demethylation pathway or fragments to nitric oxide and N-methylformaldimine as the first products of denitrosation.

Animals↗
PubMed1987

Inhibition of microsomal N-nitrosodimethylamine demethylase by diethyl ether and other anesthetics.

The inhibitory actions of diethyl ether and several other anesthetics on the metabolism of N-nitrosodimethylamine (NDMA) and other substrates were studied with rat liver microsomes. Diethyl ether was an effective inhibitor of the low Km NDMA demethylase, showing characteristics of a competitive inhibition. Inhibition of NDMA metabolism was also observed in the liver post-mitochondrial supernatant fraction prepared from ether-anesthetized rats. Selectivity in the inhibitory action of diethyl ether was demonstrated; the ether was most effective against NDMA demethylase, less potent against p-nitroanisole demethylase and N-nitrosomethylbenzylamine demethylase, and not effective against the metabolism of aminopyrine or benzphetamine. Other anesthetics such as chloroform, isoflurane, enflurane, and halothane also effectively inhibited NDMA demethylase. The work demonstrates that diethyl ether is an efficient inhibitor of NDMA metabolism by the microsomal monooxygenase systems.

Anesthesia, Inhalation↗
PubMed1987

Concurrent generation of methylamine and nitrite during denitrosation of N-nitrosodimethylamine by rat liver microsomes.

With the goal of identifying the organic amine product(s) of enzymatic N-nitrosodimethylamine (NDMA) denitrosation, 4 mM NDMA was incubated with liver microsomes from ethanol-treated rats. The concentrations of dimethylamine and methylamine were determined by derivatization with 2,4-dinitrofluorobenzene followed by gas chromatography-mass spectrometry. There was no net increase in the concentration of dimethylamine during incubation, but the yield of methylamine was equimolar with that of nitrite. Additional incubations of NDMA using acetone-induced microsomes, 1 mM and 0.1 mM substrate, gave methylamine/nitrite ratios of 0.9 and 0.7, respectively, confirming the quantitative linkage between these two products. Control incubations conducted with pure methylamine or dimethylamine indicated that the secondary amine is not a significant intermediate in the metabolic generation of the primary amine. Experiments with 15N-labeled NDMA showed that the methylamine nitrogen came from the amino moiety of the nitrosamine. The results suggest that NDMA metabolism is best viewed as a competition between at least two important pathways, demethylation and the presumably deactivating denitrosation route, a formulation which seems to account for the previously reported detection of methylamine as a urinary metabolite of NDMA and for the production of less than theoretical yields of labeled dinitrogen gas during NDMA metabolism.

Acetone↗
PubMed1987

Potential for metabolic deactivation of carcinogenic N-nitrosodimethylamine in vivo.

Enzymatic cleavage of N-nitrosodimethylamine (NDMA) to nitrite (normally representing about 10% of the total metabolism in vitro) also produces methylamine in yields roughly equimolar to those of nitrite, suggesting that the 'denitrosation' pathway may be responsible for the previously unexplained detection of methylamine as a urinary metabolite of NDMA and, at least in part, for the recovery of less than stoichiometric amounts of dinitrogen in 15N-labelling experiments. We have now followed excretion of labelled methylamine by rats receiving 14C-NDMA as a possible index of the extent of in-vivo denitrosation. Correcting for the proportion of labelled methylamine recovered in the urine following its administration under the conditions used for NDMA, 2.5-10% of the NDMA metabolism in Fischer rats appeared to proceed by a methylamine-forming route. The results are consistent with the conclusion that the metabolism of NDMA is best viewed as a competition between two pathways, with denitrosation diverting a significant proportion of the clearance to a presumably deactivating metabolic route at the expense of the activating alkylation pathway responsible for carcinogenesis.

Alkylation↗
PubMed1987

Kinetic isotope effect on the demethylation and denitrosation of N-nitrosodimethylamine in vitro.

Deuteration of N-nitrosodimethylamine (NDMA) has been shown to decrease the carcinogenicity of this compound. This result is believed to be due to a kinetic isotope effect on the metabolic activation of this carcinogen, but conflicting views exist concerning whether the isotope substitution affects the Km or Vmax of the reaction. In order to elucidate the molecular basis of these observations, as well as the mechanisms of the demethylation and denitrosation reactions, the metabolism of NDMA and deuterated NDMA (NDMA-d6) was studied using acetone-induced rat-liver microsomes. The demethylation of NDMA displayed a Km of 0.06 mM and a Vmax of 7.9 nmol/min per mg protein. Deuteration of NDMA increased the Km value by five fold but did not appreciably affect the Vmax. The denitrosation of NDMA also displayed a Km of 0.06 mM, but the Vmax was 0.83 nmol/min per mg; deuteration again increased the Kmax several fold but had no effect on the Vmax. The results indicate that deuteration inhibits the metabolism of NDMA by increasing the Km but not the Vmax and suggest that there is a close relationship between the demethylation and denitrosation reactions.

Acetone↗
PubMed1987

Organ specificity, metabolism and reaction with DNA of aliphatic nitrosomethylalkylamines.

Aliphatic nitrosomethylalkylamines are carcinogens with a remarkable organ specificity in rats, the principal targets being liver, oesophagus and bladder. We have determined the extent of DNA methylation in these tissues following a single oral dose (0.1 mmol/kg; 6-h survival) of each of 12 homologues, ranging from N-nitrosodimethylamine (NDMA, C1) to N-nitrosomethyldodecylamine (C12). Methylpurines (7- and O6-methylguanine; 7-meGua and O6-meGua) were determined by cation-exchange high-performance liquid chromatography with fluorescence detection. Highest levels of hepatic DNA methylation were found with NDMA (C1) and N-nitrosoethylmethylamine (NEMA, C2), the most potent hepatocarcinogens in this series. Concentrations of methylpurines in liver DNA decreased with increasing chain length from C1 to C5. Administration of the higher homologues (C6-C12) caused levels of DNA methylation which by themselves were considered too low to account for their hepatocarcinogenicity. In rat oesophagus, DNA methylation closely paralleled carcinogenicity, the most effective agents being the butyl and pentyl derivatives (C4 and C5). Levels of DNA methylation in bladder epithelium were close to the limit of detection (C6,C9,C10,C12) and there was no apparent correlation with carcinogenicity. It is concluded that initiation of malignant transformation by DNA methylation alone (through hydroxylation of the nitrosamine at the methylene alpha-carbon) could be operative for C1-C5. For the higher homologues, this type of DNA modification is insufficient to explain the complex pattern of tissue specificity.

Animals↗
PubMed1986

Extent of DNA 2-hydroxyethylation by N-nitrosomethylethylamine and N-nitrosodiethylamine in vivo.

At low doses, N-nitrosomethylethylamine (NMEA) selectively produces liver tumors in rats, whereas beta-trideuterated NMEA also includes esophageal carcinomas under these conditions. Since deuteration is capable of retarding enzymic hydroxylation, these studies suggest that beta-hydroxylation plays a significant role in the organ specificity of NMEA. To test the hypothesis that this metabolic pathway occurs in vivo to yield a hydroxyethylating intermediate, we have determined the extent of hydroxyethylation of hepatic DNA in male Fischer 344 rats following a single i.p. injection of [1-ethyl-14C]NMEA (6.3 mg/kg, 4 h survival). After hydrolysis in 0.1 M HCl, DNA purines were analysed by cation exchange chromatography. Of the major alkylpurines identified, 7-ethylguanine (7-etG) (6.7 mumol/mol guanine) and O6-ethylguanine (4.1 mumol/mol guanine) comprised 13 and 8% of the eluted radioactivity, respectively. 7-(2-Hydroxyethyl)guanine (7-heG) was the only hydroxyethyl adduct detectable, and comprised less than 2% of the amount of 7-etG. 3-Ethylguanine and 3- and 7-ethyladenine were also identified as products of NMEA metabolism. Similar analyses were carried out on hepatic DNA from rats treated with N-nitrosodi[1-14C]ethylamine (6.9 mg/kg, 4 h survival). Only trace amounts of 7-heG could be detected. The very low concentrations of beta-hydroxyethylated DNA bases observed suggest that this route of metabolism does not contribute significantly to the carcinogenicity of these compounds.

Alkylation↗