Biomedical subjects

M Wiessler

Publications and source records attributed to M Wiessler.

At least 73 records · Page 4Linked to original sources

PubMed1990

N-nitrosamino phosphates are unlikely transport forms for activated nitrosamines.

Some of the target organs for nitrosamine carcinogenicity have a low activating capacity but many carcinogenic nitrosamines can be activated in the liver. Conjugates, such as phosphates, are chemically accessible reaction products of 1-OH-nitrosamines, and are either potential detoxication products or potential transport forms for activated nitrosamines. 14C-labeled 1-(N-ethyl-N-nitrosamino)ethyl phosphate was tested for its ability to enter primary rat hepatocytes but no uptake was detectable. No uptake was observable into fibroblasts and human leukocytes. N-Nitrosomethylbenzylamine is efficiently 1-C-hydroxylated by hepatocytes but the corresponding 1-C-phosphate was detectable neither in the cells nor in the surrounding medium. N-Nitrosamino-1-phosphates, unlike 1-glucuronides, therefore, do not seem to be important for nitrosamine toxicokinetics.

Animals↗

PubMed1990

Peroxidase-mediated reaction of the carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxynaphthalene with transfer ribonucleic acid.

Horseradish peroxidase in the presence of hydrogen peroxide has the ability to mediate the activation of carcinogenic 1-phenylazo-2-hydroxynaphthalene (Sudan I) to DNA- and transfer RNA (tRNA)-bound products in vitro. tRNA is more accessible for modification by the activated carcinogen studied. tRNA modified by activated Sudan I becomes colored and has an absorption maximum of approximately 480 nm. Binding of metabolite(s) to tRNA is inhibited by ascorbate, glutathione, Mg2+ ions and nitrosobenzene. The mechanism of these protections was shown to be different for the different agents. tRNA modified by activated Sudan I exhibits a significantly increased acceptance for L-methionine. Enzymatic hydrolysis of modified tRNA with subsequent separation of nucleosides by HPLC suggests that the covalent modification of tRNA originating from the formation of more than one adduct with the nucleosides in tRNA is the predominant interaction of the activated Sudan I with tRNA.

Animals↗

PubMed1990

Mechanism of formation and 32P-postlabeling of DNA adducts derived from peroxidative activation of carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxynaphthalene (Sudan I).

Horseradish peroxidase in the presence of hydrogen peroxide mediates the activation of carcinogenic 1-phenylazo-2-hydroxynaphthalene (Sudan I) to DNA-bound products in vitro. The peroxidase activating system is greater than 10 times more effective with respect to DNA modification by Sudan I than the microsomal enzymes containing cytochrome P450. The DNA-binding reaction of the Sudan I metabolite(s) formed by the peroxidase system is dependent on Sudan I and H2O2 concentration and pH. Reactive intermediate(s) or product(s) of the Sudan I oxidation by peroxidase with a short half-life are responsible for the DNA modification. DNA modified by peroxidase-activated Sudan I becomes colored and has an absorption maximum at approximately 480 nm. The modification of DNA by Sudan I metabolites(s) formed by the peroxidase system is inhibited by some compounds of physiological importance (ascorbate, glutathione, Mg2+ ions) and by radical trapping agents (nitrosobenzene, methyl viologen). 32P-Postlabeling assay of the DNA modified by Sudan I activated by the peroxidase system indicates that the covalent DNA adduct formation is the principal type of the DNA modification. Four major and several minor adducts of deoxyribonucleotide 3',5'-bisphosphate from DNA with Sudan I metabolite(s) were detected by the classical Randerath 32P-postlabelling assay as well as by the nuclease P1 version of the same method.

Animals↗

PubMed1990

Influence of dithiocarbamates on the metabolism and toxicity of N-nitrosodimethylamine in rats.

Five secondary amines and secondary amino acids were reacted with carbon disulfide to yield dithiocarbamates. These compounds were tested for their influence on biochemical parameters and on some biological effects of N-nitrosodimethylamine (NDMA). The following results were found. (i) All dithiocarbamates tested reduced the activity of N-nitrosodiethylamine-deethylase and completely inhibited the N-nitrosodimethylamine-demethylase in the rat liver. A striking influence on the glutathione content and on the activity of glutathione-S-transferase and glutathione-reductase was not observed. (ii) The excretion of unmetabolized N-nitrosodimethylamine in rat urine during 24 h increased from 0.1% without pretreatment to 3.6% of the given dose on combination with a single dose of dithiocarbamate. (iii) The acute toxicity of NDMA could be reduced with diethanol-dithiocarbamate. After a single simultaneous application of the inhibitor with NDMA, the LD50 was increased from 40 to 66 mg/kg. When the sulfur compound was administered twice, both simultaneously and 24 h after the nitrosamine, lethality was almost completely inhibited. We conclude from these results that dithiocarbamates may be suitable compounds for chemoprevention of nitrosamine-induced tumors.

Animals↗

PubMed1990

Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides in DNA.

The plant extract aristolochic acid (AA) has been used as a herbal drug in many cultures since antiquity. In 1982 AA was shown to be mutagenic and a strong carcinogen in Wistar rats. The crude mixture consists of five nitrophenanthrene carboxylic acid derivatives with aristolochic acid I [AA I; 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxyli c acid] being the major component. The isolated compound has been found to be mutagenic in the Ames assay. The major metabolite of AA I formed under anaerobic conditions in vitro and excreted in vivo in several species including man, is the reduction product aristolactam I. Using the 32P-postlabeling assay, we could show that AA I forms covalent DNA adducts upon metabolic activation in vitro and in vivo in different organs in the rat. Xanthine oxidase, a mammalian nitroreductase, has served as a sufficient model system mimicking the reductive route of in vivo activation of carcinogenic nitroarenes. This paper reports on two major fluorescent adducts of AA I formed by in vitro reaction of AA I with xanthine oxidase and deoxyguanosine or deoxyadenosine. After isolation and purification by preparative HPLC the adducts were characterized by 1H-NMR, FAB mass, UV/Vis and fluorescence spectroscopy. Their structures were elucidated as 7-(deoxyguanosin-N2-yl)-aristolactam I and 7-(deoxyadenosin-N6-yl)-aristolactam I. These findings are in marked contrast to the results reported for other nitroaromatic carcinogens, where C8-modified deoxyguanosine adducts predominate and N2-substituted deoxyguanosine derivatives are found as minor reaction products. Our results suggest a cyclic N-acylnitrenium ion with delocalized positive charge as the ultimate carcinogenic species, binding preferentially to the exocyclic amino group of purine nucleotides in DNA.

Aristolochic Acids↗

PubMed1990

Pharmacokinetics of N-nitrodimethylamine and N-nitromethylamine in the rat.

Oxidative metabolism of radioactively labeled N-nitrodimethylamine in rats was compared with that of N-nitromethylamine. Within 7 h, 20% of N-nitrodimethylamine was metabolized to CO2 but only 4% of N-nitromethylamine. The poor oxidative metabolism of N-nitromethylamine is also reflected in the blood levels determined after i.v. administration to catheterized rats. N-Nitrodimethylamine was cleared rapidly from rat blood, while N-nitromethylamine was rapidly distributed into body water but had a long elimination half-life. An amount equal to 5.2% of the dose of the monomethyl compound was excreted intact in urine, but only 0.004% of the dimethyl compound. The pharmacokinetic data obtained were compared with the published data on the pharmacokinetics of the structural analog N-nitrosodimethylamine.

Animals↗

PubMed1990

Intestinal first-pass metabolism of nitrosamines. 4. Metabolism of N-nitrosodibutylamine in vascularly autoperfused jejunal and ileal loops of rats.

N-Nitrosodi-[1-14C]butylamine (NDBA) has been shown to undergo a high first-pass metabolism in isolated perfused rat small intestinal segments. Metabolites resulting from omega-hydroxylation of NDBA, the bladder carcinogens N-nitrosobutyl-(4-hydroxybutyl)amine (NB4HBA) and N-nitrosobutyl-(3-carboxypropyl)-amine (NB3CPA), accounted for greater than 90% of the total radioactivity absorbed. In the present study using vascularly perfused rat small intestinal segments, the high first-pass metabolism of NDBA could be confirmed under near in vivo conditions despite the much higher absorption rate. At the end of the 36 min experimental period 70-80% of the dose have been absorbed via the portal blood as opposed to 1-10% of the dose after 2 h in vitro perfusion. omega-Hydroxylation was again the most important metabolic pathway. However, the relationship of NB3CPA to NB4HBA was shifted in favor of NB4HBA, indicating a concentration and absorption rate dependency in the further metabolism of NB4HBA to NB3CPA.

Animals↗

PubMed1990

32P-postlabelling analysis of the DNA adducts formed by aristolochic acid I and II.

We report the quantitation of DNA adducts in target and nontarget organs of male Wistar rats treated orally with five daily doses (10 mg/kg body wt) aristolochic acid I (AAI) or aristolochic acid II (AAII), the major components of the herbal drug aristolochic acid, a forestomach carcinogen in the rat. DNA adducts were detected and analysed using the nuclease P1-enhanced variation of the Randerath 32P-postlabelling assay. The highest level of DNA adducts formed was by AAI in the target organ, forestomach (330 +/- 30 adducts/10(8) nucleotides), but high levels were also observed in a non-target tissue, the glandular stomach (180 +/- 15). Lower amounts of adducts were detected in liver, kidney and urinary bladder epithelium. With AAII the binding levels were generally lower than the AAI, the highest level of adducts being detected in kidney (80 +/- 20 adducts/10(8) nucleotides) and lower levels in liver, stomach and urinary bladder epithelia. Adduct patterns similar to those in vivo were observed in two new in vitro assays. Rat faecal bacteria were shown to be able to activate AAI and AAII to reactive species, which were trapped with exogenous calf thymus DNA and analysed by postlabelling. Incubation of AAI and AAII in explanted rat stomach held in short-term organ culture resulted in DNA adduct formation in the epithelia of both forestomach and glandular stomach. To assign the recently characterized in vitro nucleoside adducts of AAI to the bisphosphate derivatives, a new ion-pair HPLC procedure on a reversed-phase column was developed. By monitoring Cerenkov radiation on-line, a good separation of AAI adducts was observed, demonstrating that adducts formed in vivo were chromatographically indistinguishable with those formed in vitro, and previously characterized as an aristolactam I moiety bound covalently to the exocyclic amino groups of deoxyadenosine and deoxyguanosine.

Animals↗

PubMed1990

New mixed disulfides of L-cysteine derivatives and of glutathione with diethyldithiocarbamic acid and 2-mercaptoethanesulfonic acid.

It is generally assumed that thiol anticarcinogens like disulfiram (DSF) or sodium 2-mercaptoethane-sulfonate (mesna) after in vivo administration react rapidly with serum protein sulfhydryl groups forming mixed disulfides. Different methods have been established for the synthesis of mixed disulfides between cysteine or glutathione and diethyldithiocarbamate (DDTC) or mesna in order to elucidate their chemical and biological effects. A short summary is given of pilot biochemical experiments carried out with two mixed disulfides.

Antineoplastic Agents↗

PubMed1989

Metabolism of N-nitroso-hydroxyethyl-alkylamine phosphate esters in the rat.

The metabolism of 1-(N-methyl-N-nitrosamino)-ethylphosphate and 1-(N-ethyl-N-nitrosamino)-ethylphosphate in the rat was investigated. The determination of blood clearance, organ clearance, excretion of parent compounds in the urine and the exhalation of radiolabeled CO2 originating from a nitrosaminophosphate demonstrated a rapid metabolism of the compounds. The high activity of alkaline phosphatase in kidney caused a very rapid degradation of the nitrosamino phosphates in kidney homogenate, whereas the compounds were relatively stable in liver homogenate and serum. We, therefore, suggest a rapid degradation of such nitrosamino conjugates, if they are formed at all, in vivo.

Animals↗

PubMed1989

Carcinogenic properties of N-nitrodimethylamine and N-nitromethylamine in the rat.

Male and female BDVI rats were treated with 1 mmol/kg x week and 0.5 mmol/kg x week of N-nitrodimethylamine (NTDMA) or N-nitromethylamine (NTMA). NTMA induced mainly tumors of the nasal cavity (esthesioneuro-epithelioma) while NTMA induced neurinoma of the spine, spinal nerves and peripheral nerves. NTMA was slightly less potent than the dimethyl compound. Females were less susceptible than males to the carcinogenic action of the nitramines.

Administration, Oral↗

PubMed1988

Organ and cell specificity of DNA methylation by N-nitrosomethylamylamine in rats.

N-Nitrosomethylamylamine (NMAA) is a potent carcinogen in rodents with the esophagus as the principal target organ. The present study aims at an assessment of DNA methylation by NMAA in various rat tissues and an identification of cell populations actively involved in its bioactivation. Adult male F344 rats received a single i.p. dose of N-nitroso[methyl-14C]amylamine (0.1 mmol/kg). After 6 h organs were removed and the DNA was extracted, hydrolyzed in 0.1 M HCl, and subjected to radiochromatography on Sephasorb-HP. Highest levels of DNA alkylation were found in esophagus (798 mumol 7-methylguanine/mol mol guanine), followed by nasal epithelium (672 mumol) and liver (624 mumol). Trachea, lung, forestomach, and kidney had considerably lower levels of alkylation and in glandular stomach, spleen, and duodenum, values were close to the limit of detection. Specific target cell populations were identified autoradiographically and by immunohistochemistry using a rabbit antiserum to O6-methyldeoxyguanosine. In the esophagus, NMAA was selectively metabolized by the basal cells of the mucosa. In the respiratory tract, O6-methyldeoxyguanosine was almost exclusively present in the tracheal and bronchiolar epithelia. In the nasal cavity, labeled nuclei were found in both the olfactory and the respiratory epithelium and in the serous glands. Our studies indicate that NMAA and related asymmetrical nitrosamines are, in addition to liver, preferentially metabolized in tissues derived from the ventral entoderm, including the upper respiratory and gastrointestinal tract.

Animals↗

PubMed1988

DNA adduct formation of aristolochic acid I and II in vitro and in vivo.

Aristolochic acid I (AA I) and aristolochic acid II (AA II), the two main ingredients of the carcinogenic plant extract aristolochic acid (AA), are metabolized to reactive intermediates which bind covalently to DNA in vitro and in vivo. DNA adduct formation was analysed by the 32P-postlabelling assay. In in vitro incubations with rat liver 9000 g supernatant (S9) and calf thymus DNA (CT-DNA), AA I showed an identical pattern of DNA adducts on thin-layer chromatograms under aerobic and anaerobic conditions, whereas AA II gave rise to DNA adduct formation only anaerobically. The anaerobically obtained DNA adduct pattern by AA II in vitro was similar to the AA I adduct patterns. Aristolactams I and II, the metabolites of AA I and AA II formed under anaerobic conditions, did not form DNA adducts in the presence of S9 mix and CT-DNA. Incubations with xanthine oxidase, known to enzymatically reduce aromatic nitro groups, also activated AA I and AA II to reactive intermediates, producing almost identical adduct patterns as obtained by S9 mix-mediated metabolism. Activation of AA I by S9 mix in the presence of poly(dG) resulted in the formation of two adducts, one of which was shown to be chromatographically indistinguishable from an adduct obtained by reaction with CT-DNA. For the in vivo studies AA I and AA II were administered orally to male Wistar rats, and DNA from liver, brain, oesophagus, stomach lining, forestomach lining, kidney and bladder was analysed for DNA adducts by 32P-postlabelling. The adduct patterns in DNA from forestomach and kidney--target tissues of AA--and DNA from non-target tissues like stomach lining and liver were similar to the patterns obtained from the in vitro incubations. In the bladder (also a target tissue) only AA II gave rise to DNA adduct formation. These findings suggest that DNA adduct formation by AA I and AA II does not directly correlate with the initiation of the carcinogenic process and subsequent tumour formation in target tissues in the rat.

Animals↗

PubMed1988

Intestinal first-pass metabolism of nitrosamines. 2. Metabolism of N-nitrosodibutylamine in isolated perfused rat small intestinal segments.

In a previous study a high first-pass metabolism of N-nitro-sodi-[1-14C]butylamine (NDBA, 0.32-730 microM) has been shown in isolated perfused rat small intestinal segments. In the present work the identification and quantitation of metabolites in samples of perfusate and absorbed fluid (absorbate) is reported. After HPLC enrichment and purification five metabolites could be identified by GLC-MS: N-nitrosobutyl-(3-hydroxybutyl)amine (NB3HBA), N-nitrosobutyl-(4-hydroxybutyl)amine (NB4HBA), N-nitrosobutyl-(3-carboxypropyl)amine (NB3CPA), N-nitrosobutyl-(2-hydroxy-3-carboxypropyl)amine (NB2H3CPA) and N-nitrosobutylcarboxymethylamine (NBCMA), representing greater than 95% of total metabolites. omega-hydroxylation leading to NB4HBA and NB3CPA accounted for 75-95% of total metabolites. The formation of their follow-up products NB2H3CPA and NBCMA was too small for quantitative analysis. In absorbate NB3CPA was by far the most important metabolite. The hydroxylation of NB4HBA to NB3CPA was more efficient in jejunal as compared to ileal segments. omega-1-hydroxylation to NB3HBA and, as reported previously, alpha-hydroxylation were only minor metabolic pathways. In conclusion, a high first-pass metabolism of NDBA to the proximate bladder carcinogen NB3CPA takes place during its absorption in rat small intestine. This is in contrast to the observation in rat liver preparations, where alpha- and omega-1-hydroxylation are the predominant pathways. The intestinal first-pass metabolism may play a key role explaining the increased incidence of bladder tumors in rats after low oral doses of NDBA.

Animals↗

PubMed1988

Intestinal first-pass metabolism of nitrosamines. 3. Identification of metabolites of N-nitrosodiamylamine.

In a previous study a high first-pass metabolism of N-nitrosodi-[1-14C]amylamine (NDAA, 0.59-350 microM) has been shown in isolated perfused rat small intestinal segments. In the present work the identification of metabolites in samples of perfusate and absorbed fluid (absorbate) is reported. After HPLC enrichment and purification five metabolites could be identified by GLC-MS which are the result of omega- and omega-1-hydroxylation, N-nitrosoamyl-(5-hydroxyamyl)amine (NA5HAA), N-nitrosoamyl-(4-hydroxyamyl)amine (NA4HAA), N-nitrosoamyl-(4-oxoamyl)amine (NA4OAA), N-nitrosoamyl-(4-carboxybutyl)amine (NA4CBA), and N-nitroso-amyl-(2-carboxyethyl)amine (NA2CEA). In rat urine the presence of only two of these metabolites, NA4HAA and NA2CEA, has been reported in the literature.

Animals↗

PubMed1988

Influence of thiocompounds on the metabolism of N-nitrosodiethylamine.

Diethyldithiocarbaminate (DDTC), mercaptoethanesulfonate (MESNA) and ethylxanthate (PEX) were tested for their influence on N-nitrosodiethylamine (NDEA) metabolism. The exhalation rate of 14CO2 released from [14C]NDEA was decreased by PEX, but not by MESNA or DDTC. The sulfur compounds led to an increased excretion of unchanged NDEA in urine in the order PEX greater than DDTC much greater than MESNA. The activity of NDEA-deethylase in liver microsomal system was decreased only after DDTC or PEX treatment. Glutathione content and glutathione-S-transferase activity were not affected significantly by any of the tested compounds. NDEA-induced single-strand breaks in liver cell DNA were inhibited after PEX treatment.

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↗

PubMed1987

Investigation into the pharmacodynamics of the carcinogen N-nitrodimethylamine.

N-Nitrodimethylamine (NTDMA) was found to be a carcinogen of the nasal mucosa leading to aesthesioneuroepitheliomas in BDVI rats. N-Nitromethylamine (NTMA), a product of the oxidative metabolism of NTDMA, was also carcinogenic, leading to neurogenic tumours of the lumbar region of the spine. The 100,000 X g supernatant of both liver and nasal mucosa contains an enzyme capable of reducing NTDMA to N-nitrosodimethylamine (NDMA). In the microsomal fraction of both organs, NTDMA is oxidized to formaldehyde. The fractions from nasal mucosa have a higher capacity than the corresponding liver fractions to both oxidize and reduce NTDMA. NDMA was detected in blood and urine from rats treated with NTDMA. The elimination of NTDMA from blood occurs biphasically, with an initial half-life of 3.5 min.

Anaerobiosis↗