PubMed Health⌕ Search

Biomedical subjects

K E Appel

Publications and source records attributed to K E Appel.

At least 37 records · Page 2Linked to original sources
PubMed1987

Enzymatic denitrosation of diphenylnitrosamine: activation or inactivation?

Nitrosodiphenylamine was tested for induction of DNA single strand breaks in rat hepatocytes and Chinese hamster V 79 cells with the alkaline filter elution assay. While in rat hepatocytes DNA damage could be observed, negative results were obtained in V 79 cells. In view of the metabolic capacity of hepatocytes and the chemical structure of nitrosodiphenylamine, it seems likely that cytochrome P-450-dependent, reductive denitrosation might be necessary for exerting this effect. Therefore the metabolism of nitrosodiphenylamine was investigated in phenobarbital-induced mouse liver microsomes. Various metabolites were determined by HPLC. One metabolite was identified as diphenylamine, whereas the others were characterized as p-hydroxydiphenylamine and its corresponding quinoneimine. It is postulated that diphenylhydroxylamine, which is not found as a metabolite, might be involved in exerting the observed genetoxic effects.

Animals↗
PubMed1987

Metabolic denitrosation of diphenylnitrosamine: a possible bioactivation pathway.

Nitrosodiphenylamine was tested for induction of DNA single strand breaks in rat hepatocytes and Chinese hamster V 79 cells with the alkaline filter elution assay. While in rat hepatocytes DNA damage was observed, negative results were obtained in V 79 cells. In view of the metabolic capacity of hepatocytes and the chemical structure of nitrosodiphenylamine it seems likely that cytochrome P-450-dependent, reductive denitrosation might be necessary for exerting this effect. Therefore the metabolism of nitrosodiphenylamine was investigated in phenobarbital-induced mouse liver microsomes and some of the metabolites were also tested. One metabolite was identified as diphenylamine whereas the others were identified as a ring-hydroxylated derivative of diphenylamine and its corresponding quinoneimine. Diphenylhydroxylamine which was not detected in the microsomes as a metabolite produced a significant amount of DNA single strand breaks in V 79 cells. When diphenylhydroxylamine was incubated with microsomes electron spin resonance spectrum was observed which indicated the formation of the diphenylnitroxide radical. This radical seems to be mediated by auto-oxidation rather than by enzymatic catalysis. Whether diphenylhydroxylamine might be responsible for the observed genetoxic effects of nitrosodiphenylamine assumed to be produced via active oxygen species is discussed.

Animals↗
PubMed1987

Inhibition of bleomycin-induced DNA strand breaks in V 79 Chinese hamster cells by the antioxidant propylgallate.

Bleomycin induced DNA single-strand breaks in Chinese hamster V 79 cells which were detected by the alkaline filter elution assay. In the presence of propylgallate, an antioxidant, the amount of DNA single-strand breaks was significantly reduced. The production of DNA strand breaks by methylnitronitrosoguanidine used as a positive control was not influenced by propylgallate. It is suggested that propylgallate inhibits the generation of DNA single-strand breaks by trapping reactive oxygen species produced by the bleomycin-iron(II) complex.

Animals↗
PubMed1987

Some aspects of cytochrome P450-dependent denitrosation of N-nitrosamines.

The present paper deals with three aspects of cytochrome P450-dependent denitrosation of N-nitrosamines. (1) Nitrate was found in addition to nitrite as a metabolic product of the denitrosation reaction when N-nitrosamines were incubated with a microsomal system. This could also be shown when nitric oxide was added to the microsomes. (2) In order to determine the amount of denitrosation in vivo, the nitroso group of N-nitroso-N-methylaniline was labelled with the 15N isotope and administered to rats; then, the concentrations of 15N-nitrate and 15-N-nitrite in the urine were quantified by measuring the reaction of nitrate and benzene to nitrobenzene. It is estimated from these data that about 33% of the applied dose of 15N-nitroso-N-methylaniline is denitrosated in vivo. (3) Although N-nitrosodiphenylamine (NDPhA) has been classified as a noncarcinogen, recent long-term and short-term studies have cast some doubt. In order to evaluate the mechanism by which NDPhA exerts its possible genetoxic effects, its metabolism was studied in vitro, and NDPhA and its metabolites were tested for induction of DNA single-strand breaks in rat hepatocytes and in Chinese hamster V79 cells. One metabolite was identified as diphenylamine; others were suspected to be the 4-hydroxylated derivative and its corresponding quinoneimine. NDPhA caused DNA damage in rat hepatocytes but not in V79 cells. Diphenylamine also gave negative results in V79 cells, but its putative metabolite, diphenylhydroxylamine, induced a significant increase in DNA single-strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Metabolic nitrite formation from N-nitrosamines: are there other pathways than reductive denitrosation by cytochrome P-450?

To demonstrate whether there are any pathways of nitrite formation from N-nitrosamines other than reductive denitrosation by cytochrome P-450 we performed the following experiments. An esterified alpha-hydroxylated nitrosamine was incubated in a microsomal system to test if nitrite generation is coupled with or linked to the oxidative bioactivation pathway. Simultaneously, inhibitors of microsomal esterases were added to test if the intact molecule or a metabolite from the oxidative metabolism was responsible for nitrite formation. To check if the superoxide radical anion could be related to the mechanism of nitrite generation, nitrosamines were incubated with a xanthine oxidase/hypoxanthine system. To test if the OH radical was involved in nitrite formation, nitrosamines were incubated with an artificial hydroxy-radical generating system (xanthine oxidase/hypoxanthine system supplemented with Fe2+/EDTA). Measurable amounts of nitrite were detected after incubation of the esterified-hydroxylated N-nitrosamine when the hydrolysis by microsomal esterases was inhibited by diisopropylfluorophosphate or paraoxon and when the N-nitrosamines were incubated with the artificial hydroxy-radical generating system. Nitrite formation could not be detected in the O2(-)-generating system (xanthine oxidase/hypoxanthine) or when the esterified alpha-hydroxylated N-nitrosamine was incubated without inhibition of the microsomal esterases. These results demonstrate that besides reductive denitrosation by cytochrome P-450, nitrite generation from N-nitrosamines can also be caused by hydroxy-radicals. The importance of this possible pathway for the in vivo situation of nitrosamine metabolism is discussed.

Animals↗
PubMed1984

Denitrosation of diphenylnitrosamine in vivo.

A single dose of diphenylnitrosamine (NDphA) was applied orally or intraperitoneally (i.p.) to rats. The urine was sampled and analysed for nitrite/nitrate by ion chromatography and for diphenylamine (DphA) plus hydroxydiphenylamine by gas chromatography. The major metabolite was nitrate. Nitrite and DphA were found in minor amounts. In a somewhat higher concentration, a monohydroxylated DphA was detected. It is concluded that NDphA is denitrosated to nitric oxide (NO) and DphA in the organism and that NO is then converted into nitrite and nitrate.

Administration, Oral↗
PubMed1984

Metabolic inactivation of N-nitrosamines by cytochrome P-450 in vitro and in vivo.

Nitrite was formed on incubation of N-nitrosamines with both microsomal systems and a reconstituted system consisting of cytochrome P-450 and NADPH P-450 reductase from pig liver. Nitrite was not obtained when the nitrosamines were incubated with NADPH P-450 reductase alone or when molecular oxygen or NADPH was omitted. Various inhibitors of the microsomal monooxygenase decreased nitrite generation. Furthermore, nitrite and a substantially higher amount of nitrate could be found in the urine of rats given N-nitrosodiphenylamine. Diphenylamine was also detected. From in-vitro studies, it is concluded that denitrosation of N-nitrosamines is a cytochrome P-450-dependent process, which also occurs in vivo.

Animals↗
PubMed1983

Irreversible binding of acrylonitrile to nucleic acids.

1. [2,3-14C]Acrylonitrile was incubated with rat-liver microsomes, NADPH and either DNA, RNA or bovine serum albumin. Irreversible binding occurred to the macromolecular targets. Binding was lower when incubations were performed without microsomes. 2. Most of the 14C bound to DNA, RNA or polynucleotides (poly-A, poly-C, poly-G, poly-U) after incubation of [2,3-14C]acrylonitrile with rat-liver microsomes and 'conventional' re-isolation of the nucleic acids was removed from the macromolecular target when subsequently chromatographed on hydroxyapatite. 3. Radioactivity attached to DNA after prolonged non-enzymic incubations with [2,3-14C]acrylonitrile was also removed from the DNA by chromatography on hydroxyapatite. 4. When [2,3-14C]acrylonitrile was administered to rats (i.p.), incorporation of 14C into the natural bases of hepatic RNA was observed. In contrast with previous experiments with [1,2-14C]vinyl chloride, no radioactive [1-N6]etheno-adenine could be detected in RNA. 5. After administration of [2,3-14C]acrylonitrile to rats, hepatic DNA was isolated and hydrolysed by a modified enzymic procedure. Chromatography on PEI-cellulose showed two 14C peaks which did not co-chromatograph with any known standard. The amount of 14C in these presumed alkylation products was too low to allow chemical identification. 6. It is concluded that acrylonitrile, either itself or its metabolites, can alkylate nucleic acids. However, the extent of irreversible nucleic-acid binding is quantitatively much less than that observed with vinyl halides.

Acrylonitrile↗
PubMed1982

Metabolic nitrite formation from N-nitrosamines: evidence for a cytochrome P-450 dependent reaction.

Nitrite was formed on incubation of N-nitrosamines with a reconstituted monooxygenase system, consisting of cytochrome P-450 (P-450) and NADPH P-450 reductase from pig liver. Nitrite was not obtained when the nitrosamines were incubated with NADPH P-450 reductase alone or when molecular oxygen or NADPH were omitted. Interaction of nitrosamines with the reconstituted P-450 system or with hemoglobin under reducing conditions resulted in optical spectra identical with those obtained with nitrite. It is proposed that N-nitrosamines are denitrosated by electron transfer from the hemoprotein iron to the nitrosamine molecule.

Animals↗
PubMed1981

Interaction of acrylonitrile with hepatic microsomes of rats and men.

Acrylonitrile (AN) showed spectral interaction with hepatic microsomes from mouse, rat and man. Whereas human and rat liver microsomes resulted in ligand AN-binding spectra, mouse liver microsomes behaved differently. Hepatic microsomes from phenobarbital-treated mice with AN showed a type I substrate binding; ligand spectra are recorded with microsomes from benzo[a]pyrene-treated mice. Microsomes from untreated control mice showed a mixed type behaviour.

Acrylonitrile↗
PubMed1980

Carcinogen metabolism and carcinogen-induced strand breaks in DNA of isolated liver cells.

Viable rat hepatocytes were isolated and incubated for various times in suspension or monolayer. RNA and DNA synthesis and the contents of cytochromes P450 and b5, as well as microsomal functions, were studied throughout the incubation. Incubation with dimethylnitrosamine (DMN) resulted in protein and RNA alkylation that were linear with time over a period of 4 hours. Correspondingly the appearance of single strand breaks in DNA could be demonstrated following in vivo or in vitro administration of the carcinogen.

Animals↗
PubMed1980

Effect of ethanol on microsomal metabolism of dimethylnitrosamine.

Rat liver microsomes were prepared from male and female controls and from animals pretreated for 3 weeks with ethanol, and incubated with dimethylnitrosamine (DMN) and an NADPH-regenerating system. The formation of formaldehyde and nitrite as well as the alkylation of microsomal protein were found to be greatly enhanced, especially in the low DMN concentration range, as a result of long-term ethanol induction. In contrast, ethanol or tetrahydrofuran, when incubated simultaneously with DMN, inhibited microsomal metabolism of the carcinogen.

Animals↗
PubMed1980

Denitrosation of N-nitrosomorpholine by liver microsomes; possible role of cytochrome P-450.

Liver microsomes from male mice and rats were incubated with N-nitrosomorpholine (MoNA) and an NADPH-regenerating system. The formation of nitrite was measured after induction or inhibition of the microsomal monooxygenase system. Pretreatment of the animals with phenobarbital (PB) enhanced nitrite formation by about 200%, while 3-methylcholanthrene (3-MC)-induction showed no exceptional effects. Various specific inhibitors of the monooxygenase function including carbon monoxide decreased nitrite formation. In conjunction with results obtained by spectra studies it is suggested that N-nitrosomorpholine is denitrosated by a reduction process in which cytochrome (cyt.) P-450 seems to be involved. Nitricoxide formed is partly converted to nitrite under these conditions.

Aminoacetonitrile↗
PubMed1979

Influences of inducers and inhibitors of the microsomal monooxygenase system on the alkylating intensity of dimethylnitrosamine in mice.

Male mice were pretreated with various drugs, which are known either induce or to inhibit cytochrome P 450 function. Five hours after the administration of 14C-dimethylnitrosamine (10 mg/kg) in pretreated and control mice, total and specific radioactivities of cellular macromolecules of the liver were determined. Various types of nucleic acids were extracted and hydrolyzed, and the bases and nucleotides separated by ion-exchange-chromatography. Radioactivity resulting from incorporation and alkylation was measured. Pretreatment of mice with the inducers phenobarbital and 3-Methylcholanthrene decreased the alkylation rates, while administration of the inhibitors SKF 525 A and CFT 1201 caused an increase. These results appear to contradict the accepted activation mechanism of nitrosamines if DMN demethylation as catalyzed by cyt. P 450 is influenced in the same way as other cyt. P 450 dependent reactions.

Alkylation↗
PubMed1979

Influence of drugs on activation and inactivation of hepatocarcinogenic nitrosamines.

The alkylation of liver macromolecules following in vivo administration of dimethylnitrosamine (DMN) was diminished after phenobarbital or 3-methylcholanthrene induction of the microsomal monooxygenase system, but increased after SKF 525 A pretreatment. Determination of CH2O and CH3+formation in microsomal incubation systems revealed the same results for low DMN concentrations. An interpretation is given on the basis of spectrophotometric data and electron spinresonance (EPR) measurements, by proposing that metabolic inactivation of nitrosamines is catalysed by a reduction process in which cyt. P 450 seems to be involved.

Alkylation↗