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Biomedical subjects

L K Keefer

Publications and source records attributed to L K Keefer.

At least 91 records · Page 5Linked to original sources
PubMed1986

Methylation versus ethylation of DNA in target and nontarget tissues of Fischer 344 rats treated with N-nitrosomethylethylamine.

Bioactivation of N-nitrosomethylethylamine can be initiated by hydroxylation of either the methyl or ethyl moiety leading to an ethylating or methylating intermediate, respectively. This study was designed to determine which of these metabolic pathways predominates in vivo and to what extent DNA is alkylated in the target and nontarget tissues. Adult male Fischer 344 rats received a single i.p. or p.o. dose (4.4 mg/kg, 0.05 mmol/kg) of N-nitrosomethylethylamine, 14C-labeled in either the methyl or ethyl group (survival time, 4 h). DNA was analyzed by Sephasorb-HP chromatography following acid hydrolysis in 0.1 M HCl. Concentrations of 7-methylguanine in hepatic DNA were 170-200 times higher than those of 7-ethylguanine. This is approximately 2.6 times the 7-methylguanine:7-ethylguanine ratio of 68, observed when DNA is reacted in vitro with equimolar amounts of the direct alkylating agents N-nitrosomethylurea and N-nitrosoethylurea, suggesting that hydroxylation at the alpha-position of the ethyl group of N-nitrosomethylethylamine proceeds at about 2.6 times the rate as at the methyl group. Concentrations of 7-methylguanine in liver were approximately 15 times higher than in kidney, 100 times higher than in esophagus, and 200 times higher than in lung. Addition of ethanol to the drinking water (5%) caused a slight interorgan shift in metabolism with a decrease in the 7-methylguanine ratio for liver:esophagus by 50% and an increase in the 7-methylguanine ratio for liver:kidney by 40%.

Alkylation↗
PubMed1985

Use of 3,4-dichlorobenzenethiol as a trapping agent for alkylating intermediates during in vitro metabolism of nitrosamines.

Our studies using 3,4-dichlorobenzenethiol as a probe for methylating agent production during exposure of N-nitrosodimethylamine to rat liver S-9 preparations produced results different from those of an investigation reported in the literature. Methyl-3,4-dichlorophenyl thioether was detected, but the quantities found were not significantly different from the background levels of methylation product detected in the absence of nitrosamine. Only about 10% of the thioether isolated after incubating N-nitrosodi[14C]methylamine as substrate was radioactive. The results indicate that the majority of the methyl groups transferred to the sulfur nucleophile in our experiments came from components of the incubation mixture other than the nitrosamine. Some artifactual methylation was also associated with the analytical procedure. We conclude that 3,4-dichlorobenzenethiol should be used with caution in studies of alkylation during the in vitro metabolism of carcinogenic nitrosamines.

Animals↗
PubMed1985

Inhibition of N-nitrosodimethylamine metabolism in rats by ether anesthesia.

Short-term exposure to diethyl ether strongly inhibits the metabolism of N-nitrosodimethylamine (NDMA). Twenty-six 6-week-old male Fischer 344 rats were exposed to ether vapor until their righting reflex was lost (approximately 2 min). The animals were removed from the ether and NDMA was immediately administered by i.v. bolus injection at a dose of 300 microgram/kg via a cannula surgically inserted 20 h earlier. A second group of 28 rats received injections of NDMA in an identical manner but without ether exposure. In the unanesthetized animals blood levels of NDMA declined with a half-life of 11 min; by contrast essentially constant blood levels of NDMA were observed in ether-treated animals for 120 min after removal from the anesthetic. The apparent total systemic clearance for the 5-h experiment was reduced from 43 ml/min/kg without ether to 5 ml/min/kg with ether. Diethyl ether has been found previously to inhibit the metabolism of other drugs requiring oxidative metabolism but the suppression of clearance documented here appears to be unusually pronounced. It is recommended that ether's potential for altering metabolic rates be carefully considered when planning or interpreting animal experiments.

Anesthesia↗
PubMed1985

Low-dose in vivo pharmacokinetic and deuterium isotope effect studies of N-nitrosodimethylamine in rats.

The rates of elimination of N-nitrosodimethylamine (NDMA) and its fully deuterated analogue (N-nitrosodi[2H6]methylamine, [2H6]NDMA) were studied in vivo to explore the origins of the difference in their carcinogenicity. Male Fischer 344 rats, 7.5 weeks of age, were given nitrosamine bolus doses of 1.35 mumol/kg by tail vein injection and 2.02 or 4.05 mumol/kg by p.o. gavage. Animals were sacrificed at various time points from 2.5 to 180 min after i.v. administration or 5 to 120 min after p.o. dosage, and their blood was analyzed for NDMA by gas chromatography-high resolution mass spectrometry. After i.v. injection, blood nitrosamine concentrations declined in an apparently biexponential manner with a terminal half-life of 10 min for NDMA and 12 min for [2H6]NDMA. The apparent total systemic blood clearances for NDMA and [2H6]NDMA were 39 and 26 ml/min/kg, respectively. The apparent steady-state volumes of distribution were nearly identical (297 and 309 ml/kg, respectively). The areas under the curve after 2.02- and 4.05-mumol/kg p.o. doses were proportional to dose. The apparent bioavailability of NDMA was 8%, while that of [2H6]NDMA was 21%. Isotope effects calculated as the ratios of first-pass metabolism, total systemic clearances, bioavailabilities, and intrinsic hepatic clearances were 1.2, 1.5, 2.6, and 3.2, respectively. The isotope effect determined from blood concentrations measured after simultaneous administration of NDMA and [2H6]NDMA by steady-state infusion (each at 1.5 mumol/kg/h) was 2.6 +/- 0.9 (SD). This study thus provides quantitative reference data on the time course of the disappearance of both N-nitrosodimethylamine and its deuterated analogue from blood (over 5 to 8 half-lives) after doses similar to those used to elicit liver tumors in chronic feeding studies, confirms the first-pass effect on their metabolism using direct blood measurements, and permits estimation of their bioavailabilities from actual blood concentrations. The results suggest that elimination pathways not involving alpha-hydroxylation are more important than is currently recognized.

Animals↗
PubMed1984

Destruction of carcinogenic and mutagenic N-nitrosamides in laboratory wastes.

The chemical degradation of five N-nitrosamides used widely for the experimental induction of cancer has been studied with the goal of identifying, and experimentally validating, reliable methods that can be recommended for the destruction of carcinogenic N-nitrosoureas and related compounds in laboratory wastes. Although data are not yet complete, preliminary evidence indicates that none of the five methods studied thus far is ideal for hazard-control purposes. Decomposition with 1 mol/L potassium hydroxide solution destroyed the N-nitrosamides, but generated diazoalkanes, which are carcinogenic, toxic and potentially explosive. Treatment with strong acid in the presence of sulfamic acid or iron filings completely decomposed all N-nitrosamides without forming diazoalkanes, but failed in the presence of solvents which were immiscible with water. Cleavage with hydrogen bromide in glacial acetic acid proceeded to a point of maximum degradation, following which gradual reformation of the N-nitrosamide was observed; this resynthesis could be avoided by carefully bubbling nitrogen through the reaction mixture, but degradation was slow or failed completely in the presence of hydroxylic solvents. Permanganate oxidation was effective in sulfuric acid solution, but was incomplete when an alcohol or dimethyl sulfoxide was present. Salmonella typhimurium tester strains TA1535, TA1530 and TA100, which detect base-pair substitutions in DNA, detected mutagenic degradation products in each of the destruction methods, with the exception of the hydrobromic acid/acetic acid procedure.

Carcinogens↗
PubMed1983

Reducing nitrosamine contamination in cutting fluids.

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.

Catalysis↗
PubMed1983

Safe disposal of carcinogenic nitrosamines.

A simple one-step procedure for chemically degrading nitrosamine residues generated in the research laboratory is described. Treatment with aluminum-nickel alloy powder and aqueous alkali rapidly reduced all 11 nitrosamines studied to the corresponding amines. Hydrazines were produced as transitory intermediates, but these potential carcinogens were also easily reduced under the conditions employed, and no products except amines, ammonia, and, in some cases, alcohols were detected in the final reaction mixtures. Reduction proceeded smoothly in every other solvent system tested, except that reactions in acetone or dichloromethane solution were sometimes slow, incomplete, and/or led to unidentified products; therefore, we cannot recommend the procedure for use in these solvents. Otherwise, the method was efficient, reliable, and inexpensive and has been recommended as one of the preferred means of degrading potentially carcinogenic nitrosamines to innocuous products. Details of its application to some decontamination and disposal problems commonly encountered in the research laboratory are provided. Data illustrating this procedure's advantages over six other reducing systems are also presented.

Chemical Phenomena↗
PubMed1983

Deuterium isotope effect on metabolism of N-nitrosodimethylamine in vivo in rat.

The maximal rates of metabolic oxidation of N-nitrosodimethylamine (NDMA) and N-nitrosodimethylamine-d6 (NDMA-d6) in vivo (VH and VD, respectively) have been measured by following 14CO2 exhalation in rats after intraperitoneal injection of the two 14C-labelled carcinogens at high doses (20 or 40 mg/kg). Complete deuteration of NDMA reduced only slightly the maximal rate of metabolism when the two substrates were administered separately (VH/VD approximately 1.2). However, much larger (approximately 4-fold) deuterium isotope effects were observed when mixtures of NDMA with NDMA-d6 were injected. These results are tentatively interpreted as evidence that C-H bond cleavage is not a rate limiting feature of overall metabolism, but that the complex between NDMA and the principal enzyme(s) metabolizing it in vivo freely equilibrates with unbound substrate. Single, large, intraperitoneal doses of NDMA and NDMA-d6 produced a similar alkylation of rat liver DNA and also of kidney DNA. However, a small oral dose (54 micrograms/kg) of NDMA-d6 produced 1/3 less alkylation of liver DNA and 3 times as much alkylation of kidney DNA as did an equimolar dose of NDMA. The reduction in alkylation of liver DNA correlates well with, and possibly explains, the decreased ability of NDMA-d6 to induce liver tumors in rats. The associated increase in the alkylation of kidney DNA suggests that this change is due to a decrease in the amount of nitrosamine removed from the portal blood on the first pass through the liver.

Animals↗
PubMed1983

Carcinogenesis and aging. II. Modifying effect of aging on metabolism of methyl(acetoxymethyl)nitrosamine and its interaction with DNA of various tissues in rats.

Young (3 month-old) and old (14 month-old) female outbred rats received a single i.p. dose (13 mg/kg) of N-[14C]methyl-Nacetoxymethylnitrosamine [( 14C]DMN-OAc), which, under these conditions, selectively induces intestinal tumours. Complete DMN-OAc breakdown occurred within 30 min in both young and old rats but exhalation of 14CO2 continued for over 1 h in young rats and over 3 h in old rats. The highest level of methylation in both young and old rats was found in the DNA of epithelial cells of the colon and in other adjacent abdominal organs (liver and uterus). The initial capacity for excision of the O6-methylguanine from liver DNA was greater in young animals, but further this DNA adduct was repaired more efficiently by the liver of old rats. In the DNA of ileal and colonic enterocytes, O6-methylguanine excision was higher in young than in old animals. The DNA tertiary structure, measured by the sedimentation pattern of nucleoids in neutral sucrose gradient, was damaged in old rats, and, to a lesser extent, in young rats. The non-uniform pattern of DNA damage in young and old animals may be associated with differing carcinogenic effects of DMN-OAc in rats of different ages, a hypothesis which is currently under test.

Aging↗
PubMed1982

N-nitrosation of secondary amines by nitric oxide via the 'Drago complex'.

Diethylamine (DEA) and nitric oxide (NO) form a stable 1:1 complex (DEA/NO) which can be oxidized to N-nitrosodiethylamine (NDEA). Spectroscopic data for the complex are consistent with the ionic structure originally proposed by Drago & Paulik (1960). Attempts to confirm this structure by X-ray crystallography are continuing. Oxidation of DEA/NO to NDEA appears to be effected by an acid-catalysed dissociation to DEA and NO, followed by autoxidation of NO to an N-nitrosating higher oxide of nitrogen.

Chemical Phenomena↗
PubMed1980

Inhibitory agents and chemical and mechanisms in the dihalomethane-mediated nitrosation of amines with solid nitrite.

Methylene chloride and certain other dihaloalkanes react efficiently with dissolved secondary amines and, in presence of insoluble sodium nitrite, yield N-nitroso compounds. Mechanistic studies indicate that the intermediates in the nitrosation mixture include the formaldiminium ion, the amine hydrochloride and the diaminomethane (aminal). They also indicate that the formaldiminium ion is by far the most reactive toward sodium nitrite, which probably reacts with the former by nucleophilic attack. Ascorbic acid, ascorbyl palmitate, dehydroascorbic acid, potassium iodide and potassium carbonate inhibited the reaction, whereas alpha-tocopherol, Trolox and sodium ascorbate were found to be ineffective. Ascorbic acid reacted with the secondary amine in a complex fashion. The mode of inhibition probably does not involve reduction of the solid nitrite.

Chemical Phenomena↗
PubMed1980

The sequential determination of nitrite, N-nitroso compounds and nitrate and its application.

The selective determination of nitrite, N-nitroso compounds as a group and nitrate can be accomplished directly on most food or other matrices by quantifying the nitric oxide released following the additions in sequence of acetic acid, hydrogen bromide and titanous chloride under an atmosphere of nitrogen. Most of the many other compounds potentially formed from nitrite in a biological system are either decomposed to nitric oxide in the refluxing solvent system alone or else do not yield nitric oxide at any stage throughout the determinations of nitrite and N-nitroso compounds. Only nitrolic acids and S-nitrothiols, like nitrosamines and nitrosamides, require the use of hydrogen bromide for breakdown to nitric oxide. The application of the present procedure to the determination of nitrite and N-nitroso compounds (as a group) in a series of animal diets has shown the presence of levels of nitrite below the limit of detection of more conventional methods, even in those diets not deliberately treated with nitrite. Evidence was also obtained for the presence of non-volatile, non-extractable N-nitroso compounds in diets both treated and untreated with high levels of nitrite. Confirmation was provided in the case of one diet not treated with nitrite, in that it was found to convert morpholine into its N-nitroso derivative, which was detected by gas chromatography with a Thermal Energy Analyzer detector.

Animals↗
PubMed1980

Metal complexes as promoters of N-nitrosation reactions: a progress report.

Our research on three classes of transition metal-containing N-nitrosating agents is described. One class, the nitrito complexes, can give rise in organic solvents to quantitative yields of N-nitrosamines on exposure to secondary amines. The second class is capable of activating nitrite as an N-nitrosating agent in aqueous alkaline media; it involves N-coordination of nitrite, followed by removal of oxide ion to form the active metal nitrosyl species. The third class of N-nitrosating agent involves an as yet incompletely characterized metal complex containing a ligand having the formula NO3. The possible significance of compounds in these classes in environmental or in vivo N-nitrosamine formation is discussed.

Chemical Phenomena↗
PubMed1978

Focal suppression and induction of hyperplasia by the bladder carcinogens butyl(4-hydroxybutyl)nitrosamine and buty(3-carboxypropyl)nitrosamine in organ-cultured rat bladder epithelium.

The effects of the bladder carcinogens butyl(4-hydroxybutyl)nitrosamine (BBN) and butyl(3-carboxypropyl)-nitrosamine (BCPN) on proliferating transitional rat epithelium in organ culture were studied. At low to intermediate concentration ranges (0.5--2.9 mM), both compounds appeared to stimulate hyperplasia in some regions of epithelia. The major effect of both carcinogens, however, was to suppress hyperplasia in other regions of epithelia and, at higher concentrations (5--6 mM), to cause necrosis. For comparable concentrations, BBN was more effective in suppressing proliferation and causing necrosis than was BCPN.

Animals↗