Michael D. Threadgill

The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinones in isolated hepatocytes

In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity.

Differences between rodents and humans in the metabolic toxification of N,N-dimethylformamide☆

The widely used industrial solvent N,N-dimethylformamide (DMF) causes liver damage in occupationally exposed persons and is suspected of involvement in the generation of certain occupational malignancies. Here the extent of the biotransformation of DMF to three urinary metabolites has been compared in humans and rodents. The metabolites, which were quantified by gas chromatography (GC) are N-(hydroxymethyl)-N-methylformamide (HMMF), which yielded N-methylformamide on GC analysis, a species which decomposed to formamide on GC analysis, and N-acetyl-S-(N-methylcarbamoyl) cysteine (AMCC), measured after derivatization with ethanol to give ethyl N-methylcarbamate. Ten volunteers who absorbed between 28 and 60 mumol/kg DMF during an 8-hr exposure to DMF in the air at 60 mg/m3 excreted in the urine within 72 hr between 16.1 and 48.7% of the dose as HMMF, between 8.3 and 23.9% as formamide, and between 9.7 and 22.8% as AMCC. AMCC, together with HMMF, was also detected in the urine of workers after occupational exposure to DMF. The portion of the dose (0.1, 0.7, or 7.0 mmol/kg given ip) which was metabolized in mice, rats, or hamsters to HMMF varied between 8.4 and 47.3% of the dose; between 7.9 and 37.5% were excreted as formamide and only between 1.1 and 5.2%, as AMCC. The results suggest that there is a quantitative difference between the metabolic pathway of DMF to AMCC in humans and rodents. It is argued that the hepatotoxic potential of DMF may be linked to the extent of its metabolic conversion to AMCC.

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.

S-(N-methylcarbamoyl)-N-acetylcysteine: a urinary metabolite of the hepatotoxic experimental antitumour agent N-methylformamide (NSC 3051) in mouse, rat and man

Abstract N-Methylformamide (NMF, NSC 3051) is an antineoplastic agent in mice[1,2]. In clinical trials in which the potential of NMF for the therapy of human cancers was evaluated, manifestations of liver damage were observed[3,4,5].The mouse appears to be particularly sensitive to the hepatotoxic properties of NMF[6,7] and results of mechanistic studies in mice suggest that a reactive metabolite of NMF is responsible for its hepatotoxicity[7.8]. Whereas NMF is metabolised in vitro only to a very minor extent by liver fractions or isolated mouse hepatocytes, it undergoes extensive metabolism in vivo in rodents[9]. Carbon dioxide, methylamine and N-hydroxymethylformamide have been identified as major metabolites of NMF[10].In that study, a further metabolite was detected but not characterised. We now report the identification of a new urinary metabolite of NMF and suggest that its precursor(s) may well be responsible for the hepatotoxicity and/or the antitumour activity of NMF.

Activities of serine hydroxymethyltransferase in murine tissues and tumours

The specific activity of the enzyme serine hydroxymethyltransferase (EC 2.1.2.1) was determined in various murine, rat and human tumour cell lines. The activities of the enzyme were also investigated in tissues of non-tumour bearing DBA/2 mice and BALB/c mice bearing the PC6 ascites tumour. The highest enzyme activity in the murine tissues was found in the liver and then the kidneys. The enzyme was present in all the tissues assayed. The activities of enzyme found in the tumours varied considerably, with the PC6 ascites, Walker 256 and Lewis lung cells, being the highest.

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.

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.

Cyclisation of a Substituted N-Acetylserine to A 2-Methyldihydrooxazole With Dimethylaminosulphur Trifluoride

Abstract Treatment of N-acetyl-2-(benzyloxymethyl)serine methyl ester with dimethylaminosulphur trifluoride resulted unexpectedly in cyclisation to methyl 4-(benzyloxymethyl)-2-methyl-4,5-dihydro-1,3-oxazole-4-carboxylate.

The Reaction of Diethanolamine with 2-Chloronitrobenzene-A Reinvestigation

Abstract In a report on the reaction of 2-chloronitrobenzene (1) with diethanolamine (2), Meltsner et al 1 claim that the expected SNAr product, N-(2-nitrophenyl)diethanolamine (3), is not formed; rather that the products are 2,2′-dichloroazobenzene (4), 2-nitrophenol (5), 2-chloroaniline (6) and 4-(2-aminophenyl)morpholine (7). Similar products in which the nitro function is reduced are also reported2 for the corresponding reaction with ethanolamine. In this laboratory, in an attempted preparation of 2,2′-dichloroazobenzene (4) for reference purposes in photochemical studies on the antineoplastic agent 5-(3-azido-4-chlorophenyl)-6-ethyl-pyrimidin-2,4-diamine3, the expected SNAr product (3) was obtained along with other products.

Butylation of 2-Methylnaphthalene-1,4-dione (Menadione) by Solvent Sulpholane During Radical Methylation and Ethylation

Abstract Treatment of 2-methylnaphthalene-1, 4-dione with potassium peroxydisulphate, silver nitrate and acetic or propanoic acids in aqueous sulpholane gave 3-butyl-2-methylnaphthalene-1, 4-dione in addition to the expected 3-alkyl-2-methylnaphthalene-1, 4-diones.