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Biomedical subjects

A Gescher

Publications and source records attributed to A Gescher.

At least 91 records · Page 5Linked to original sources
PubMed1988

Glutathione depletion in the guinea pig and its effect on the acute cochlear toxicity of ethacrynic acid.

There is controversy as to whether or not the acute cochlear toxicity of ethacrynic acid (EA) is dependent upon its metabolic conversion to EA-cysteine via conjugation with glutathione. In order to investigate this we examined the acute effects of EA on cochlear potentials in guinea pigs in which glutathione levels were decreased by prior administration of (+/-)-buthionine sulphoximine (BSO), an inhibitor of glutamylcysteine synthetase. First, we determined the effects of BSO on hepatic and renal glutathione levels in the guinea pig. Guinea pigs (pigmented animals of both sexes or male albino animals) were killed at intervals up to 72 hr after i.p. administration of 1.6 g kg-1 BSO. Livers, and also kidneys in the case of pigmented guinea pigs, were removed and total glutathione (GSH + GSSG) measured. Glutathione levels reached a nadir in the liver at 24-48 hr (11% of control) and in the kidneys at 24 hr (14% of control) after administration of BSO. Hepatic but not renal levels approached control values by 72 hr. There were no sex or strain differences. Pigmented guinea pigs were anaesthetised and their endocochlear potential and a.c. cochlear potential in response to a 4 kHz tone were measured using an intracochlear microelectrode. The depression of these potentials by i.v. administration of 60 mg kg-1 EA was not affected by administration of 1.6 g kg-1 BSO 24 hr earlier, despite profound depletion of glutathione. Also prior p.o. administration of N-acetyl-L-cysteine did not affect hepatic glutathione levels nor modify the toxicity of EA. These results suggest that the acute cochlear toxicity of EA is not altered by glutathione depletion, a finding which argues against a role for the metabolic activation of EA in its ototoxicity.

Animals↗
PubMed1988

Effects of N-methylformamide on the growth, cell cycle, and glutathione status of murine TLX5 lymphoma cells.

The growth of the murine TLX5 lymphoma is inhibited in vivo by administration of N-methylformamide (Gescher, A., et al., Br. J. Cancer, 45: 843-850, 1982). Continuous incubation of TLX5 murine lymphoma cells in vitro with N-methylformamide for 72 h, at concentrations of between 43 and 170 mM (0.25 and 1% v/v), brought about a concentration-dependent decrease in growth rate (50% inhibitory concentration = 68 mM) and viability. Cell replication was decreased by 37% after 48 h exposure to 106 mM N-methylformamide, while viability was maintained at 82%. Analysis of the distribution of these cells in the cell cycle by flow cytofluorimetry showed a 23% increase in the proportion of G1 cells and a fall in the proportion of cells in the S and G2/M phases. As the drug concentration and time of exposure to N-methylformamide were increased, with an associated reduction in cell replication and viability, the proportion of G1 cells rose. When TLX5 cells were washed free of N-methylformamide after an exposure to 106 mM for 48 h and cultured in drug-free medium, the cells returned to exponential growth and to a normal cell cycle distribution. Clonogenic assays showed that the recovery of proliferation, after removal of the drug, was due to that of all those cells which, in a parallel experiment, excluded the dye trypan blue. It is concluded that the cessation of replication and the accumulation of cells in G1 of the cell cycle, after treatment with N-methylformamide, are probably not events representative of terminal differentiation but rather of cytostasis, which was accompanied by rapid cell death. Coincident with the reduction of TLX5 cell proliferation caused by N-methylformamide and the accumulation of cells in G1, cellular glutathione concentrations fell by 80%. A similar fall was induced by treatment of the cells with D,L-buthionine[S,R]-sulfoximine (5 microM) for 48 h, but this treatment had no effect on cell growth.

Animals↗
PubMed1988

Cytotoxicity and metabolism of the hepatotoxin N-methylformamide and related formamides in mouse hepatocytes.

Some N-alkylformamides such as N-methylformamide (NMF) possess hepatotoxic properties in vivo. To study the mechanism of this toxicity, suspensions of mouse hepatocytes were tested as an in vitro model system suitable for the study of the relationship between (i) the toxic potential of formamides, (ii) their metabolism to N-alkylcarbamoylating species, and (iii) their ability to deplete hepatic glutathione pools. The effects of NMF were compared with those of its analogs N-ethylformamide (NEF), N,N-dimethylformamide (DMF), formamide (F), N-methylacetamide (NMA), and N-methyldeuteroformamide ([2H]NMF). Only NEF and [2H]NMF share with NMF the ability to cause liver damage in vivo in mice. Hepatocellular toxicity was determined by measuring LDH leakage into the extracellular medium; metabolism to N-alkylcarbamoylating species was measured by GLC after derivatization with propanol to form propyl N-alkylcarbamate; glutathione concentrations were determined spectrophotometrically. Of the formamide analogs studied, only NMF and NEF caused cytotoxicity, being apparently equipotent. NMF, NEF, and [2H]NMF gave rise to the formation of detectable levels of N-alkylcarbamoylating metabolites and depleted glutathione pools. Toxicity, metabolism, and glutathione depletion were dependent on NMF concentration. [2H]NMF was markedly less cytotoxic than NMF, yielding only 35% of the amount of N-methylcarbamoylating metabolite compared to NMF and caused less depletion of glutathione than did NMF. These results parallel closely the in vivo hepatotoxic potential of NMF and its analogs, their metabolism to urinary S-(N-alkylcarbamoyl)mercapturates and their ability to deplete hepatic glutathione in mice. The results provide support for the contention that metabolism is involved with formamide-induced hepatotoxicity and suggest that suspensions of isolated mouse hepatocytes are an appropriate in vitro model for the further study of the mechanism by which formamides cause toxicity.

Acetylcysteine↗
PubMed1988

Medicinal azides. Part 3. The metabolism of the investigational antitumour agent meta-azidopyrimethamine in mouse tissue in vitro.

1. The experimental antitumour agent meta-azidopyrimethamine is deactivated by reduction of the azide moiety to yield meta-aminopyrimethamine. This reaction was followed by h.p.l.c. subsequent to incubation of the drug with homogenates prepared from liver, kidney, spleen, intestine and heart of mice. Reducing activity was highest in liver homogenate and was time dependent. Following the incubation of 530 nmol meta-azidopyrimethamine with liver homogenate equivalent to 1 g liver for 30 min under air, 84% of the agent was reduced to meta-aminopyrimethamine. 2. Reducing activity was markedly lower in incubations containing subcellular fractions obtained from liver homogenate. Under aerobic conditions the 700 g and 12,500 g supernatant fractions were able to reduce 57% and 23%, respectively, of the amount of meta-azidopyrimethamine initially present. The respective pellets possessed weak reducing ability, but on reconstitution of the supernatants with their corresponding pellets most of the reducing activity as observed in the whole homogenate was recovered. 3. Microsomes and mitochondria reduced the drug only when incubations were performed in the presence of nitrogen but not under air. 4. A fraction of the reducing activity measured in the tissue homogenates was non-enzymatic, as heat-inactivated homogenates retained less than 10% of the activity exhibited by the untreated homogenates. Neither bovine serum albumin nor glutathione could reduce meta-azidopyrimethamine under the conditions of the tissue incubations, whereas dithiothreitol was a powerful reductant. The mixed enzymatic and non-enzymatic nature, the tissue distribution and the diffuse subcellular localisation of meta-azidopyrimethamine reducing activity resemble features of the bioreduction of N-oxides.

Animals↗
PubMed1987

Hepatotoxicity of N-methylformamide in mice--I. Relationship to glutathione status.

In order to investigate the link between hepatotoxicity caused by N-methylformamide (NMF) and its ability to deplete hepatic glutathione experiments were conducted in three strains of mouse which differ in their susceptibility towards NMF-induced liver damage. NMF toxicity was measured by changes in plasma levels of sorbitol dehydrogenase and alanine and aspartate transaminases. In BALB/c mice, the most susceptible strain, a hepatotoxic dose of NMF (200 mg/kg) caused a depletion of hepatic glutathione to 21% of control levels 2 hr after drug administration. In CBA/CA and BDF1 mice the same dose of NMF depleted glutathione to 53% of control levels and did not cause hepatotoxicity. In BALB/c mice depletion of hepatic glutathione by pretreatment with buthionine sulfoximine decreased the hepatotoxic dose threshold of NMF from 150 mg/kg to 100 mg/kg. Conversely, pretreatment of mice with cysteine or N-acetylcysteine protected against both glutathione depletion and NMF-induced hepatotoxicity. The results are in accordance with the suggestion that the hepatotoxicity of NMF is associated with its metabolism to an intermediate which reacts with glutathione.

Alanine Transaminase↗
PubMed1987

Hepatotoxicity of N-methylformamide in mice--II. Covalent binding of metabolites of [14C]-labelled N-methylformamide to hepatic proteins.

Incubation of the hepatotoxin N-methylformamide (NMF) labelled either in the methyl group (OHCNH14CH3) or the formyl group (OH14CNHCH3) with mouse hepatic microsomes in the presence of NADPH, but not in its absence, led to covalent binding of metabolites to microsomal proteins. When [14C]NMF was injected into BALB/c mice radioactivity was found to be associated with liver and, to a much lesser extent, with kidney proteins. Association of radioactivity derived from OHCNH14CH3 with hepatic proteins was higher in BALB/c mice than in CBA/CA mice and in these it was higher than in BDF1 mice. Association of label derived from either isotopomer was significantly reduced but not abolished by pretreatment of mice with cycloheximide suggesting both covalent binding and metabolic incorporation of NMF metabolites. Depletion of hepatic glutathione by pretreatment of mice with buthionine sulfoximine or diethyl maleate prior to administration of OH14CNHCH3 enhanced the association of label with hepatic proteins measured 1 hr after drug injection. Covalent binding of [14C]NMF to hepatic microsomes in vitro was abolished in the presence of glutathione. It is argued that the generation of the toxic lesion and the association of NMF metabolites with hepatic proteins may be causally related even though certain mechanistic and enzymatic details of this link remain obscure.

Animals↗
PubMed1987

N-methyl antitumour agents. A distinct class of anticancer drugs?

This article reviews the structure-activity characteristics, mode of action, pharmacokinetics and clinical utility of a group of chemically dissimilar antitumour agents which have as a common structural feature the N-methyl moiety. The importance of this feature is shown by the fact that molecules without a substituent on the nitrogen or compounds with N-alkyl groups other than methyl are usually inactive in experimental systems. This observation is supported by structure-activity studies with N-alkyl derivatives of s-triazines, triazenes, formamides, hydrazines and nitrosoureas. Representatives of these structural types which have found clinical application are, respectively, hexamethylmelamine, dacarbazine, N-methylformamide, procarbazine and streptozotocin. Mode of action studies have shown that dacarbazine, procarbazine and streptozotocin can give rise to species capable of methylating nucleic acid. This may be the lesion which produces antitumour activity. The mechanism of action of N-methylmelamines and N-methylformamide remains unclear. There is good evidence that, with the exception of N-methylnitrosoureas, host metabolism is prerequisite for activity with these agents. Although not pronounced, the clinical activity of N-methyl antitumour agents is useful, particularly as activity is not associated with severe haematological toxicity. Furthermore, responses may be observed in patients resistant to bifunctional alkylating agents. It is concluded that the drugs reviewed herein show a degree of coincidence in terms of their biological properties which may warrant a common classification. The term N-methyl antitumour agent is proposed.

Alkylating Agents↗
PubMed1987

Metabolism of N-methylformamide in mice: primary kinetic deuterium isotope effect and identification of S-(N-methylcarbamoyl)glutathione as a metabolite.

S-(N-Methylcarbamoyl)glutathione has been identified by cesium ion liquid secondary ion mass spectrometry as a biliary metabolite in mice of the experimental antitumor agent and hepatotoxin N-methylformamide. Metabolism of N-methylformamide to urinary methylamine, urinary N-acetyl-S-(N-methylcarbamoyl)-cysteine and biliary S-(N-methylcarbamoyl)glutathione was found to be subject to large intermolecular primary kinetic isotope effects when hydrogen was replaced by deuterium in the formyl group (kH/kD = 5.5 +/- 0.2, 4.5 +/- 1.0 and 7 +/- 2, respectively), as shown by mass spectrometry of derivatives of these metabolites. These values indicate the existence of a common metabolic precursor for each of these metabolites. In particular, methylamine is shown not to arise from simple enzymatic hydrolysis of N-methylformamide but is associated with an oxidative process. Therefore, it is highly likely that N-methylformamide is oxidized and conjugated to form S-(N-methylcarbamoyl)glutathione which is metabolized further to N-acetyl-S-(N-methylcarbamoyl) cysteine. Either of these thiocarbamates could be hydrolyzed to give the parent thiol and the observed metabolic end products, methylamine and carbon dioxide. The presence of deuterium in the formyl moiety of N-methylformamide reduced markedly the hepatotoxicity of the compound, as shown by measurements of the activities of appropriate hepatic enzymes in plasma.

Acetylcysteine↗
PubMed1987

An investigation of the relationship between the hepatotoxicity and the metabolism of N-alkylformamides.

The hepatotoxicity and metabolism of the following close analogs of the hepatotoxic antitumor agent N-methylformamide (NMF) were investigated in CBA/CA mice: N-ethylformamide (NEF), dimethylformamide (DMF), formamide and N-methylacetamide (NMA). Apart from NMF only NEF was potently hepatotoxic as measured by the elevation of plasma activities of the enzymes sorbitol dehydrogenase and alanine and aspartate aminotransferases 24 hr after drug administration. In freeze-dried urine samples of mice which had received NEF or NMF, but not in the case of DMF, formamide or NMA, thioesters were detected by thin-layer chromatography. Evidence based on high-pressure liquid chromatography analysis and 400 MHz 1H-NMR and mass spectrometry suggests that the thioester metabolite of NEF is S-(N-ethylcarbamoyl)-N-acetylcysteine. It has been shown previously that NMF is metabolized to S-(N-methylcarbamoyl)-N-acetylcysteine. NEF also underwent extensive metabolism to ethylamine; similarly NMF was biotransformed to methylamine. In contrast, the urine of mice which had received DMF contained only very small amounts of dimethylamine and methylamine could not be detected as a metabolite of NMA. Instead, the major metabolite of NMA was identified by 400 MHz 1H-NMR spectrometry as N-(hydroxymethyl)acetamide. DMF is known to undergo extensive metabolism to its N-hydroxymethyl derivative. The results suggest that two metabolic pathways of N-alkylformamides can be distinguished: Hydroxylation at the alpha-carbon of the N-alkyl group and oxidation of the formyl moiety. The former pathway presumably constitutes a detoxification route, and the latter may well be associated with hepatotoxicity, and affords a glutathione conjugate, excreted in the urine as a mercapturate.

Acetamides↗
PubMed1986

The formation and metabolism of N-hydroxymethyl compounds--IX. N-(acetoxymethyl)-4-chlorobenzamide: an electrophile but not a mutagen in Salmonella typhimurium.

The electrophilic properties of 4-chloro-N-(hydroxymethyl)benzamide as a model compound of carbinolamides formed during the metabolic oxidation of N-methylamides were investigated. 4-Chloro-N-(hydroxymethyl)benzamide did not react with nucleophiles such as cyanide or glutathione under physiological conditions. In contrast, N-(acetoxymethyl)-4-chlorobenzamide yielded the cyanomethylamide with KCN and S-(4-chlorobenzamidomethyl)glutathione with glutathione. Under nonaqueous conditions, N-(acetoxymethyl)-4-chlorobenzamide reacted avidly with ethanethiol, with methanol and with diethylamine in the presence of base, whereas 4-chloro-N-(hydroxymethyl)benzamide did not afford products under these conditions. These results show clearly that N-(acetoxymethyl)-4-chlorobenzamide is the precursor of reactive electrophilic methyleneimines. 4-Chloro-N-(hydroxymethyl)benzamide was not biotransformed to such electrophilic species when incubated with mouse hepatic microsomes or a microsomal supernatant with acetyl-CoA or a PAPS generating system. Neither 4-chloro-N-(hydroxymethyl)benzamide nor its acetate ester were mutagenic in the short term bacterial assay using Salmonella typhimurium. Nevertheless, esters of carbinolamides, such as N-(acetoxymethyl)-4-chlorobenzamide, might possess toxic or carcinogenic properties.

Animals↗
PubMed1986

Identification by proton NMR of N-(hydroxymethyl)-N-methylformamide as the major urinary metabolite of N,N-dimethylformamide in mice.

Urine samples from mice which had received N,N-dimethylformamide were investigated by high field 1H-NMR spectroscopy. The most prominent signals in the N-CH3 region had chemical shifts identical with those of N,N-dimethylformamide (delta 2.85, 3.01) and N-(hydroxymethyl)-N-methylformamide (delta 2.91, 3.05). Resonances downfield of delta 7.5 (from formyl protons) also coincided with those of the reference formamides. When [14C]methyl-labelled N,N-dimethylformamide was injected and urine samples investigated by radio thin layer chromatography, the major area of radioactivity corresponded to the Rf of N-(hydroxymethyl)-N-methylformamide. Dimethylamine and methylamine were found to be minor metabolites of N,N-dimethylformamide.

Animals↗
PubMed1986

Comparison of metabolism and activity of an aryldimethyltriazene and an aryldiethyltriazene.

The antitumoral activity and metabolism of 1-(4-acetylphenyl)-3,3-dimethyltriazene [pAc-(CH3)2] and 1-(4-acetylphenyl)-3,3-diethyltriazene [pAc-(C2H5)2] were studied in mice. pAc-(CH3)2 showed significant antitumoral activity against M5076 ovarian reticular cell sarcoma, L1210 leukemia, EL 4 lymphoma in mice, but not against Lewis lung carcinoma. pAc-(C2H5)2 was inactive in all these murine tumors and was much more toxic than pAc-(CH3)2. pAc-(CH3)2 and pAc-(C2H5)2 were rapidly metabolized in vitro and in vivo to their respective monoalkyltriazenes and to 4-aminoacetophenone (pAc-NH2). In vitro, 79% of the dimethyltriazene was metabolized to its monomethyl analogue, but only 27% of the diethyltriazene was metabolized to the monoethyltriazene. The monoalkytriazenes were almost completely biotransformed to pAc-NH2 by a 9000 g liver fraction. The metabolic pattern in the in vitro study was comparable to that found in vivo.

Animals↗
PubMed1986

Structural studies on bioactive compounds. 4. A structure-antitumor activity study on analogues of N-methylformamide.

A series of derivatives of N-methylformamide (NMF), an experimental antitumor agent, has been prepared, having the general formula R3C(X)NR1R2 where R1 = H, CH3, CD3, CH2CF3, CH2CH2Cl, cyclopropyl, C2H5, CH2OH, CH2OR, CH2N(CH3)2; R2 = H, CH3; R3 = H, CF3, CCl3, CH3, Ph, NHCH3, N(CH3)2; and X = O, S, NH. A further short series of "push-pull" olefins of the general formula R1R2C = CHNR3R4 has been synthesized where R1 = H, CH3 and R2 = H, NO2, CN, CHO, CH3 and R3 = H and R4 = H, CH3, morpholino. These compounds have been tested for activity against the M5076 ovarian sarcoma and the TLX5 lymphoma in mice. NMF was by far the most potent agent of both series with activity against both tumors. Some other compounds showed weak activity, but there is a rigorous structural requirement for activity and most analogues were inactive. Certain members of the series exist as equilibrium mixtures of rotamers about the amide or pro-amide bonds as shown by NMR.

Animals↗