Studies of the metabolism and the toxicity of N-Methylformamide and related amides

The hepatotoxicity of the industrial solvent and investigational anti-tumour agent N-methylformamide (NMF, HOCNHCH3) and several structural analogues was assessed in mice. NMF and its ethyl analogue (NEF) were equipotent hepatotoxins causing extensive centrilobular necrosis and damage to the gall bladder. Pretreatment of mice with SKF525A did not influence the toxicity of these N-alkylformamides. Replacement of the formyl hydrogen of NMF with deuterium or methyl significantly reduced its hepatotoxicity. An in vitro model for the study of the toxicity and metabolism of N-alkylformamides was developed using isolated mouse hepatocytes. The cytotoxicity of NMF in vitro was concentration-dependent with maximal toxicity being achieved at concentrations of 5mM or above. The cytotoxic potential of related amides correlated well with their in vivo hepatotoxic potential. Pretreatment of mice with buthionine sulphoximine (BSO), which depleted hepatocytic levels of glutathione to 15% of control values, exacerbated the cytotoxicity of NMF towards the hepatocytes. NMF (1mM or above), incubated with isolated mouse hepatocytes, depleted intracellular glutathione levels to 26% of control values within 4h. Depletion of glutathione was quantitatively matched by the formation of a carbamoylating metabolite. Metabolism was dependent on the concentration of NMF and was drastically reduced in incubations of hepatocytes isolated from mice pretreated with BSO. The carbamoylating metabolite, S-(N-methylcarbamoyl)-glutathione (SMG), was identified in vitro using FAB-MS. The generation of SMG was subject to a large primary H/D kinetic isotope effect when the formyl hydrogen was replaced with deuterium. Likewise, glutathione depletion and metabolite formation were reduced or abolished by the deuteration or methylation of the formyl moiety of NMF. NEF, like NMF, depleted hepatocytic glutathione levels and was metabolised to a carbamoylating metabolite. Radioactivity derived from 14C-NMF and 14C-NEF, labelled in the alkyl moieties, was found to be irreversibly associated with microsomal protein on incubation in vitro. Binding was dependent on the presence of NADPH and was mostly abolished in the presence of reduced glutathione. SKF525A failed to influence the binding.

The role of metabolism in the toxicity of N-alkylformamides

Using ionspray tandem mass spectrometry the glutathione conjugate SMG was identified as a biliary metabolite of DMF in rats (0.003% of a dose of 5OOmg/kg DMF i.p.). Formation of this metabolite was increased five fold after induction of CYP2E1 by acetone, and was inhibited to 20% of control values following pretreatment with disulfrram. Generation of SMG from DMF in vivo was shown to exhibit a large kinetic deuterium isotope effect (KWKD=10.1 ± 1.3), which most likely represents the product of 2 discrete isotope effects on N-demethylation and formyl oxidation reactions.The industrial solvent N,N-dimethylformamide (DMF) and the investigational anti-tumour agent N-methylformamide (NMF) cause liver damage in rodents and humans. The hepatotoxicity of N-alkylformamides is linked to their metabolism to N-alkylcarbamic acid thioesters. The enzymatic details of this pathway were investigated. Hepatocytes isolated from BALB/c mice which had been pretreated with acetone, an inducer of the cytochrome P-450 isozyme CYP2E1, were incubated with NMF (10mM). NMF caused extensive toxicity (> 90% ) as determined by lactate dehydrogenase (LDH) release, compared to cells from untreated animals. Incubation of liver cells with NMF for 6 hrs caused 60±17% LDH release whilst in the presence of DMSO (10mM), an alternative substrate for CYP2E1, LDH release was reduced to 20±10% . The metabolism of NMF to S-(N-methylcarbamoyl)glutathione (SMG) was measured in incubates with liver microsomes from mice, rats or humans. Metabolism of NMF was elevated in microsomes isolated from rats and mice pretreated with acetone, by 339% and 183% respectively. Pretreatment of animals with 4-methylpyrazole induced the metabolism of NMF to 280% by rat microsomes, but was without effect on NMF metabolism by mouse microsomes. The CYP2E1 inhibitors or alternative substrates diethyl dithiocarbamate (DEDTC), p-nitrophenol (PNP) and dimethyl sulphoxide (DMSO) strongly inhibited the metabolism of NMF in suspensions of rat liver microsomes, at concentrations which did not effect aminopyrine N-demethylation. The rate of metabolism of NMF to SMG in human microsomes correlated (r> 0.8) with the rate of metabolism of chlorzoxazone, a CYP2E1 probe. A polyclonal antibody against rat CYP2E1 (10mg/nmol P-450) inhibited NMF metabolism in microsomes from rats and humans by 75% and 80% , respectively. The amount of immunoblottable enzyme in human microsomes, determined using an anti-rat CYP2E1 antibody, correlated with the rate of NMF metabolism (r> 0.8). Purified rat CYP2E1 catalysed the generation of SMG from NMF. Formation of the DMF metabolite N-hydroxymethyl-N-methylformamide (HMMF) in incubations with rat liver microsomes was elevated by 200% following pretreatment of animals with acetone. Co-incubation with DEDTC (100μM) inhibited HMMF generation from DMF by 88% . Co-incubation of DMF (10mM) with NMF (1mM) inhibited the formation of SMG by 95% . A polyclonal antibody against rat CYP2E1 (10mg/nmol P-450) inhibited generation of HMMF in incubates with rat and human liver microsomes by 68.4% and 67.5% , respectively. Purified rat CYP2E1 catalysed the generation of HMMF from DMF. Using ionspray tandem mass spectrometry the glutathione conjugate SMG was identified as a biliary metabolite of DMF in rats (0.003% of a dose of 5OOmg/kg DMF i.p.). Formation of this metabolite was increased five fold after induction of CYP2E1 by acetone, and was inhibited to 20% of control values following pretreatment with disulfrram. Generation of SMG from DMF in vivo was shown to exhibit a large kinetic deuterium isotope effect (KHKD=10.1 ± 1.3), which most likely represents the product of 2 discrete isotope effects on N-demethylation and formyl oxidation reactions.

Radical intermediates in chloroform reactions over triphenylphosphine-protected Au nanoparticles

Reactions of chloroform over triphenylphosphine-protected Au nanoparticles have been studied using electron paramagnetic resonance (EPR) spectroscopy and a spin trapping technique. Two competing reactions, abstraction of hydrogen and halogen atoms, were identified. The hydrogen abstraction reaction showed an inverse kinetic isotope effect. Treatment of nanoparticles with oxidizing or reducing reagents made it possible to tune the selectivity of radical formation from halogen to hydrogen (deuterium) abstraction. Treatment with PbO2 promoted the deuterium abstraction reaction followed by the loss of nanoparticle activity, whereas treatment with NaBH4 regenerated the nanoparticle activity towards Cl atom abstraction. X-ray photoelectron spectroscopy showed an increased Au:P ratio upon treatment with oxidizing reagents. This is likely due to the oxidation of some phosphine ligands to phosphine oxides which then desorb from the nanoparticle surface. © 2009 The Royal Societ of Chemistry.