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.

Folate metabolism in the female weanling rat

The metabolism of a mixture of [2-14C] and [3',5',7,9-3H] folic acid was studied in female weanling rats. Intact folates and folate catabolites were excreted in the urine. Folate polyglutamates were found in the tissues. Rats treated with the oestrogen diethylstilbestrol and 17 -ethynyloestradiol exhibited marked changes in the metabolic handling of folic acid and folate catabolism was greatly increased compared to controls. Allopurinol treatment gave greater label retention in the gut, with a substantial increase in catabolism compared to normals. A dose response relationship was illustrated between allopurinol dose and folate catabolism. After lead acetate dosing there was little radioactivity in the urine and tissues over 72h and more radioactivity was retained in the faeces compared to normals. Excretion of intact folates was depressed, especially 5MeTHF and 10CHOTHF. A tenfold increase in both lead and folic acid dosage resulted in an even further decrease of radioactivity in the tissues and urine over 72h. Excretion in the faeces was further elevated. Ferrous sulphate administration resulted in increased catabolism. The retention of radioactivity in the liver, kidney and gut was greatly reduced. A new method of folate analysis; Sephadex LH-20 was introduced. In vitro superoxide anion formation was illustrated using an allopurinol/xanthine oxidase system. Histological studies were employed to qualitatively and quantitatively illustrate the oxidative status in livers and brains of allopurinol and ferrous sulphate dosed rats. Increased dose related formazan deposition was observed when livers of pretreated animals were incubated with nitroblue tetrazolium. Formazan deposition was reduced in pretreated animals also treated with the anti-oxidants vitamin E, mannitol or 4-hydroxy-methyl-4,6-ditertiary-butylphenol. A possible route of folate catabolism is scission by a non-enzymic oxidation involving active oxygen species.

Aluminium hydroxide stabilised MnFe2O4 and Fe3O4 nanoparticles as dual-modality contrasts agent for MRI and PET imaging

Magnetic nanoparticles (NPs) MnFe2O4 and Fe3O4 were stabilised by depositing an Al(OH)3 layer via a hydrolysis process. The particles displayed excellent colloidal stability in water and a high affinity to [18F]-fluoride and bisphosphonate groups. A high radiolabeling efficiency, 97% for 18F-fluoride and 100% for 64Cu-bisphosphonate conjugate, was achieved by simply incubating NPs with radioactivity solution at room temperature for 5min. The properties of particles were strongly dependant on the thickness and hardness of the Al(OH)3 layer which could in turn be controlled by the hydrolysis method. The application of these Al(OH)3 coated magnetic NPs in molecular imaging has been further explored. The results demonstrated that these NPs are potential candidates as dual modal probes for MR and PET. In vivo PET imaging showed a slow release of 18F from NPs, but no sign of efflux of 64Cu. © 2014 The Authors.