The generation of potentially toxic, reactive iminium ions from the oxidative metabolism of xenobiotic N-alkyl compounds.
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Altretamine↗
Biomedical subjects
A Gescher
Publications and source records attributed to A Gescher.
N-Methylformamide (NSC 3051): a potential candidate for combination chemotherapy.
N-Methylformamide (NMF) was found to be non-toxic to the bone marrow as reflected in the absence of leukopenia in mice, even when the marrow had been compromised by prior administration of cyclophosphamide. Thus recovery from the leukopenic nadir after 160 mg/kg of cyclophosphamide was unaffected by 200 mg/kg X 10 of NMF. This combination, given to animals bearing the M5076 sarcoma, proved to have an additive antitumour effect as measured by tumour growth delay and was superior to the antitumour effect of two doses of cyclophosphamide, a regime which prolonged the leukopenia. Furthermore, the hepatotoxicity of NMF was not augmented by the addition of cyclophosphamide. When hepatotoxicity was induced in BALB/c mice bearing the NMF-resistant ADJ/PC6A plasmacytoma, cyclophosphamide fully maintained its antitumour effect. The results show NMF to be a highly specific antiproliferative agent with potential for use in the therapy of patients with a compromised bone marrow and/or in combination chemotherapy.
Animals↗
Studies on the toxicity of the antitumour agent N-methylformamide in mice.
Aspects of the toxicology of N-methylformamide (NMF), an investigational antitumour agent, were studied in mice. After injection of NMF at its LD10 (800 mg/kg) dosage the total peripheral white blood cell and platelet counts were unchanged in BALB/c mice. A mild granulocytosis was seen in this strain after administration of the LD50 (2300 mg/kg) dosage. Plasma activity of the enzyme sorbitol dehydrogenase in BDF1 mice was markedly increased after either a single injection of not less than 800 mg/kg or a chronic treatment of not less than 400 mg/kg/day over 5 days indicating the drug to be hepatotoxic. Plasma activities of L-alanine and L-aspartate aminotransferases were also increased after the chronic treatment. Chronic administration of NMF was less hepatotoxic than single dose administration of the same total dose and also increased the antitumour efficacy of NMF against the M5076 sarcoma. These results indicate that the maximum therapeutic benefit of NMF might be obtained by the use of chronic schedules and that the drug is not myelosuppressive.
Alanine Transaminase↗
Characterization of the growth inhibition induced by tumor-promoting phorbol esters and of their receptor binding in A549 human lung carcinoma cells.
Exposure of A549 human lung carcinoma cells to 10(-8) M 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a change in cell morphology and caused the arrest of cell growth. After 4-5 days of exposure to TPA the cells started to proliferate again. However, on removal of the cells from the culture flask and reseeding, the cells had regained their sensitivity towards TPA. Cells which were subcultured in the presence of 10(-8) M TPA for 9 weeks were permanently refractory to the growth-inhibitory properties of TPA. Incubation of A549 cells with [3H]phorbol-12,13-dibutyrate ([ 3H]PDB) showed that the cells possess specific phorbol ester receptors. Exposure of the cells to 10(-7) M PDB preceding the receptor binding assay led rapidly to a decline in the binding of 6 nM [3H]PDB, in case of preincubation for 24 h to 38% of the binding in cells not pre-exposed to PDB. The receptor binding capacity after pretreatment with PDB was only weakly decreased in the cells which were desensitized towards the TPA-induced growth inhibition. Thus the decrease in receptor binding on exposure to phorbol esters does not appear to cause the refractoriness of the cells towards the effect of TPA. It seems more likely that this decrease in binding capacity is part of the events by which phorbol esters cause inhibition of cell growth.
Caenorhabditis elegans Proteins↗
Oxidative properties of 12-O-tetradecanoylphorbol-13-acetate-stimulated human blood monomorphonuclear leukocytes and their toxicity against a human lung carcinoma cell line.
Human monomorphonuclear leukocytes (MMNs) stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) were found to be toxic towards human A549 lung carcinoma cells which have been desensitized against the direct growth-inhibitory effect of TPA. This toxicity was dependent on the TPA concentration and the ratio of MMNs to A549 cells. Using a TPA concentration of 10(-7) M and an effector:target cell ratio of 30:1, experiments were performed to give clues as to the mechanisms by which TPA-stimulated MMNs cause toxicity. Levels of the endogenous thiol glutathione were reduced by 37% in MMNs exposed to TPA for 24 h, but the glutathione levels in the A549 target cells were not markedly affected by TPA-stimulated MMNs. The supernatant of incubations of MMNs with TPA contained a species which was capable of oxidizing the thiol agent 5-thio-2-nitrobenzoic acid. Within 2 h, 9 nmol of this oxidant were produced by 10(7) MMNs. The oxidant exhibited a half-life of 20 h, and its formation was abolished by adding catalase (150 units/ml), azide (1 mM), or cyanide (1 mM) to the incubations of MMNs with TPA. The addition of superoxide dismutase (100 units/ml) enhanced oxidant formation. These results indicate that its generation was dependent on the myeloperoxidase:H2O2:halide system. Large amounts of an oxidizing species with properties identical to those described here have been characterized recently in polymorphonuclear leukocytes [S. J. Weiss, M. B. Lampert, and S. T. Test. Science (Wash. DC), 222: 625-627, 1983]. The toxicity exerted by TPA-stimulated MMNs was partially inhibited by superoxide dismutase and by retinoic acid (30 microM) but not at all by catalase, azide, or cyanide. Therefore, the 5-thio-2-nitrobenzoic acid oxidant does not appear to be involved in the process which led to cytotoxicity by TPA-stimulated MMNs in A549 cells.
Cell Line↗
Investigation of the mechanism of hepatotoxicity of N-methylformamide in mice: effects on calcium sequestration in hepatic microsomes and mitochondria and on hepatic plasma membrane potential.
N-Methylformamide is an antitumour drug with hepatotoxic properties. Three potential targets for hepatocellular toxic lesions caused by N-methylformamide were investigated: the mitochondrial and microsomal Ca2+ pumps and the functional integrity of the plasma membrane. The administration of N-methylformamide to mice caused a dramatic decrease in the ability of the liver mitochondria to sequester [45Ca2+]. This effect was dose-dependent and was not caused by dimethylformamide, N-hydroxymethylformamide or formamide. The microsomal Ca2+ pump was not affected by N-methylformamide. Incubations of isolated mitochondria with N-methylformamide for 1 hr also led to the inhibition of the Ca2+ sequestration. Incubation of isolated mouse hepatocytes with N-methylformamide did not cause changes in plasma membrane potential as measured using the lipophilic cation triphenylmethylphosphonium. Of the three targets studied, the mitochondrial Ca2+ pump may be the one through which N-methylformamide triggers the events leading ultimately to hepatic necrosis.
Animals↗
Studies of the mode of action of antitumour triazenes and triazines-V. The correlation of the in vitro cytotoxicity and in vivo antitumour activity of hexamethylmelamine analogues with their metabolism.
Experiments were conducted to ascertain whether the antitumour activity of hexamethylmelamine analogues correlated with their in vitro cytotoxicity and metabolism. Two analogues, namely pentamethylmelamine (PMM) and 2,2,4,4-tetramethylmelamine (TMM), and hexamethylmelamine (HMM) itself were shown to be active towards the murine ADJ/PC6A (PC6) plasmacytoma; another three, 2-chloro-4,6-bis(dimethylamino)-1,3,5-triazine (CBDT), 2,4-bis-(dimethylamino)-6-hydrazino-1, 3,5-triazine (HBDT) and 2,4,6-trimethylmelamine (TriMM) were inactive against the same tumour. The cytotoxicity of these compounds was examined against a PC6 tumour cell line in vitro. In the absence of liver microsomal activation only CBDT proved to be significantly cytotoxic at a concentration of 5 mM. In the presence of murine liver microsomes the three active antitumour agents were all cytotoxic at this concentration whereas HBDT and TriMM remained non-toxic. The degree of cytotoxicity correlated with the extent of metabolism for these analogues. The products of biotransformation of these compounds were stable precursors of formaldehyde (presumably N-hydroxymethyl intermediates) (FP) rather than formaldehyde itself. After injection of these 6 compounds to Balb/c mice the levels of FP generated in the plasma were markedly greater for the three active antitumour agents than for the inactive analogs. No free formaldehyde was detected in the plasma after administration of any of the compounds. These results suggest that for these compounds in vitro cytotoxicity correlates with in vitro biotransformation and their antitumour activity correlates with plasma levels of FP generated by metabolism in vivo.
Altretamine↗
An investigation of the mechanism of hepatotoxicity of the antitumour agent N-methylformamide in mice.
N-Methylformamide (NMF) has been reported to cause liver damage in animals and man. This hepatotoxicity was characterized in BALB/c mice by the release of liver enzymes into the plasma and by histopathological examination of livers after single and repeated administration of NMF. Whereas plasma levels of sorbitol dehydrogenase were elevated dramatically 24 hr after 400 mg/kg given as a single dose, the glutathione content of the livers was not different from controls even after repeated administration. Liver damage was apparent on gross inspection and was defined as periacinar necrosis on histopathology. A dose of 100 mg/kg did not cause damage even after repeated injections on five consecutive days. The hypothesis that NMF is metabolized to a chemically reactive species was tested. Incubation of mouse hepatocytes with 7 mM NMF for 80 min produced a decrease in intracellular glutathione. Exposure of hepatocytes to NMF for 240 min led to the production of breakdown products of lipid peroxides at levels significantly above controls. However, incubation of microsomes or mitochondria with NMF and NADPH did not lead to raised levels of lipid peroxides. The effects described were specific to NMF as incubation of N,N-dimethylformamide, N-hydroxymethylformamide or formamide with hepatocytes did not result in glutathione depletion or increased lipid peroxidation. NMF undergoes extensive metabolism in vivo and the results indicate that NMF forms a chemically reactive metabolite, even though incubation of the drug with liver fractions or hepatocytes did not lead to metabolites at levels which were analytically identifiable.
Animals↗
The chemosensitivity of a new experimental model--the M5076 reticulum cell sarcoma.
The M5076 reticulum cell sarcoma is a murine tumour of potential value in experimental chemotherapy. Experiments were conducted to ascertain the growth characteristics and chemosensitivity of this neoplasm in the BDF1 mouse. The intramuscular tumour proved to be responsive to the alkylating agents, nitrosoureas, procarbazine, DTIC and treosulphan, yet insensitive to the antimetabolites and only weakly responsive to adriamycin. Analogues of the antitumour agents hexamethylmelamine and N-methylformamide were tested against this neoplasm. The patterns of activity determined for these analogues against this tumour were identical to those previously reported against other model systems.
Altretamine↗
The formation and metabolism of N-hydroxymethyl compounds--IV. Cytotoxicity and antitumour activity of N-hydroxymethylformamide, a putative metabolite of N-methylformamide (NSC 3051).
Experiments were conducted to test the hypothesis that N-hydroxymethylformamide (HMF) is the active metabolite of the antitumour agent N-methylformamide (NMF). In an in vitro bioassay against the TLX5 lymphoma HMF was more cytotoxic than NMF; this cytotoxicity was abolished by preincubating the TLX5 cells with semicarbazide, a formaldehyde trapping agent. Similarly, the inhibition of incorporation of radiolabelled thymidine, uridine, formate and leucine into TLX5 cells elicited by HMF was eliminated by preincubation of the cells with semicarbazide. HMF is considerably less toxic to tumour-bearing BDF1 mice than NMF and, unlike NMF, does not reduce hepatic glutathione levels in vivo. HMF has no inhibitory activity against the TLX5 lymphoma or the Sarcoma 180 in mice in vivo and only marginal activity against the M5076 reticulum cell sarcoma; these tumours are highly sensitive to NMF. However, like NMF, HMF inhibits growth of the human mammary tumour MX-1 implanted in the subrenal capsule of mice.
Animals↗
Studies of the metabolism of dimethylformamide in mice.
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Animals↗
The formation and metabolism of N-hydroxymethyl compounds--III. The metabolic conversion of N-methyl and N,N,-dimethylbenzamides to N-hydroxymethyl compounds.
The stability of metabolically-generated N-(hydroxymethyl) compounds was investigated using a series of N-methylbenzamides as model substrates. N-(Hydroxymethyl)-benzamide was characterized as a major metabolite of N-methylbenzamide in vitro, and was also identified as a urinary metabolite of N-methylbenzamide. N-(Hydroxymethyl) compounds were also found as metabolites of 4-chloro-N-methylbenzamide and 4-t-butyl-N-methylbenzamide in vitro. Thus substitution in the 4-position of the phenyl ring of derivatives of N-(hydroxymethyl)-benzamide did not affect their stability sufficiently to cause degradation to formaldehyde under the conditions used. N-(Hydroxymethyl)-N-methylbenzamide was identified as a metabolite of N,N-dimethylbenzamide in vitro. However, N-(hydroxymethyl)-N-methylbenzamide was less stable than N-(hydroxymethyl)-benzamide under alkaline conditions. Furthermore, N-(hydroxymethyl)-N-methylbenzamide, unlike N-(hydroxymethyl)-benzamide and its 4-substituted derivatives, was positive in the colorimetric assay for formaldehyde, presumably because of its degradation to produce formaldehyde. Thus substitution on the nitrogen atom which bears the methyl group in N-methylbenzamide markedly affected the stability of the N-methylol produced during oxidative metabolism. N-Formylbenzamide was identified as a metabolite of N-methylbenzamide in suspensions of mouse hepatocytes and also in vivo. The mechanism for its production probably involves the generation of N-(hydroxymethyl)-benzamide.
Animals↗
The metabolism of a stable N-hydroxymethyl derivative of a N-methylamide.
N-Formylbenzamide and benzamide were characterised by high pressure liquid chromatography and mass spectrometry as products of the metabolism of N-hydroxymethylbenzamide in incubation mixtures with mouse liver preparations and isolated hepatocytes. This biotransformation occurred predominantly in 9000g and microsomal supernatant fractions and was also catalyzed by horse liver alcohol dehydrogenase fortified with NAD and could be inhibited by pyrazole. Unlike N-hydroxymethylbenzamide, which is very stable, N-formylbenzamide degraded rapidly to benzamide in buffer at pH 7.4 with a half-life of 7.8 min. The instability of N-formylbenzamide and the time course of its metabolic generation together with benzamide suggest that benzamide is a chemical breakdown product of N-formylbenzamide. N-Formylbenzamide was also tentatively identified as a urinary metabolite of N-hydroxymethylbenzamide. This is the first time that an N-hydroxymethyl compound has been shown to undergo metabolism either in vitro or in vivo.
Animals↗
Metabolism of the anticancer agent 1-(4-acetylphenyl)-3,3-dimethyltriazene.
High pressure liquid chromatography was used in combination with mass spectrometry to confirm that the main products of in vitro metabolism of 1-(4-acetylphenyl)-3,3-dimethyltriazene are 1-(4-acetylphenyl-3-methyltriazene and 4-aminoacetophenone. In addition a novel metabolite, 1-[4-(1-hydroxyethyl)-phenyl]-3,3-dimethyltriazene, possessing antitumour activity similar to the parent drug, was identified.
Animals↗
The formation and metabolism of N-hydroxymethyl compounds--I. The oxidative N-demethylation of N-dimethyl derivatives of arylamines, aryltriazenes, arylformamidines and arylureas including the herbicide monuron.
The metabolism of the N-methyl moieties of aryldimethylamines and N-methyl compounds of the general formula Aryl-X-N(Me)2, where X is either -N=N-(3-aryl-1, 1-dimethyltriazenes). -NHCO- (N'-aryl-N,N-dimethylureas) or -N=CH- (N'-aryl-N,N-dimethylformamidines) was studied using mouse liver microsomes. Products of microsomal metabolism were reincubated with mouse liver homogenate devoid of microsomes and assayed colourimetrically for formaldehyde. This allows metabolically generated formaldehyde to be distinguished from formaldehyde precursors. Whereas the N-methyl moieties of the aryldimethyltriazenes, formamidines and amines were metabolised to formaldehyde, the aryldimethylureas formed stable formaldehyde precursors upon metabolism. The products of metabolism of one such aryldimethylurea, the herbicide monuron (N'-(4-chlorophenyl)-N, N-dimethylurea) were investigated using a high pressure liquid chromatographic method. Two metabolites were found on incubation of monuron with microsomes, one of which was identified as the N-desmethyl compound by mass spectrometry. The other product showed chromatographic properties similar to 4-chlorophenylurea but resembled the monomethylaryl urea on mass spectral analysis. It is concluded that this metabolite is likely to be N'-(4-chlorophenyl)-N-hydroxymethyl-N-methylurea. A urinary product of conjugative metabolism obtained after the administration of monuron to mice also gave the mass spectrum of the monomethyl compound after deconjugation which suggests that a conjugated N-hydroxymethyl compound may have been formed in vivo.
Amines↗
Studies of the mode of action of antitumour triazenes and triazines--IV. The metabolism of 1-(4-acetylphenyl)-3,3-dimethyltriazene.
The metabolism of 1-(4-acetylphenyl)-3,3-dimethyltriazene has been studied in vivo and in vitro in mice. This dimethyltriazene was extensively metabolised in vivo and HPLC analysis of the plasma revealed the presence of two metabolites, the monomethyltriazene, 1-(4-acetylphenyl)-3-methyltriazene, and the arylamine, 4-aminoacetophenone. The dimethyltriazene was also biotransformed in vitro by a 9000 g fraction of mouse liver homogenate to products which were selectively toxic to TLX5 lymphoma cells. HPLC analysis of the products of in vitro metabolism under these conditions showed the presence of the monomethyltriazene but in an amount insufficient to account for the observed cytotoxicity. The monomethyltriazene was itself rapidly biotransformed by a 9000 g fraction of mouse liver homogenate, and by isolated mouse hepatocytes.
Animals↗
Studies of the mode of action of antitumour triazenes and triazines-III. Metabolism studies on hexamethylmelamine.
These is good evidence that the antitumour agent hexamethylmelamine (HMM) undergoes oxidative metabolic activation which might occur in the liver and/or extrahepatically. The hepatic microsomal N-methylmelamine metabolizing enzymes were investigated in mice and exhibited different affinities for different melamine derivatives. The apparent Km values are 0.09 mM for HMM, 0.23 mM for pentamethylmelamine, 0.91 mM for 2,2,4,6-tetramethylmelamine and 1.7 mM for trimethylmelamine. HMM inhibited its own metabolism in vitro at substrate concentrations greater than 0.05 mM. Its hepatic microsomal N-demethylation rate was reduced when the mice were pretreated with the hepatic glutathione depleting agent methyliodide. Injection of hexaethylmelamine, a derivative of HMM without antineoplastic properties against the M5076 sarcoma in mice, lead to plasma concentrations of drug and metabolite pentaethylmelamine which were only a fraction of the drug and metabolite levels achieved after a similar dose of HMM.
Altretamine↗
Assessment of renal function during high-dose cis-platinum therapy in patients with ovarian carcinoma.
Five courses of cis-dichlorodiammine platinum (II) (100 mg/m2) were given to 22 patients with advanced stage III and IV ovarian cancer. Renal function was assessed by measurement of creatinine clearance, urinary osmolality and urinary B2-microglobulin (B2MG) in all patients, and by urinary alanine aminopeptidase (AAP) and N-acetyl-B-glucosaminidase (NAG) excretion in seven patients. Serum creatinine, creatinine clearance, urinary osmolality, and urinary B2-microglobulin were within the reference ranges and did not change significantly after five courses of cis-platinum in any patient. There was a significant increase in the urinary excretion of both enzymes (AAP and NAG) within 2 days of cis-platinum administration (NAG P less than 0.05 and AAP P less than 0.07). There was evidence of a cumulative effect during treatment for AAP (P less than 0.025).
Acetylglucosaminidase↗