Investigation of the metabolism of N, N-Dimethylformamide and its metabolites

The industrial solvent N, N-dimethylformamide (DMF) causes liver damage in humans. The hepatotoxicity of N-alkylformamides seems to be linked to their metabolism to N-alkylcarbamic acid thioesters. To clarify the role of metabolism in DMF hepatotoxicity, the metabolic fate of DMF was investigated in rodents. DMF was rapidly metabolised and excreted in the urine as N-hydroxymethyl-N-methyl-formamide (HMMF), N-acetyl-S-(N-methylcarbamoyl) cysteine (AMCC) and a metabolite measured as formamide by GLC. At high doses (0.7 and 7.0mmo1/kg) a small proportion of the dose was excreted unchanged. AMCC, measured by GLC after derivatisation to ethyl N-methylcarbamate, was a minor metabolite. Only 5.2% of the dose (0.1mmo1/kg) in rats or 1.2% in mice was excreted as AMCC. The minor extent of this metabolic pathway in rodents might account for the marginal liver damage induced by DMF in these species. In a collaborative study, volunteers were shown to metabolise DMF to AMCC to a greater extent than rodents. Nearly 15% of the inhaled dose (0.049mmo1/kg) was excreted as AMCC. This result suggests that the metabolic pathway leading to AMCC is more important in humans than in rodents. Consequently the risk associated with exposure to DMF might be higher in humans than in rodents. The metabolism of formamides to S-(N-alkylcarbamoyl) glutathione, the metabolic precursor of the thioester mercapturates, was studied using mouse, rat and human hepatic microsomes. The metabolism of NMF (10mM) to S-(N-methylcarbanoyl)glutathione (SMG) required the presence of GSH, NADPH and air. Generation of S-(N-methyl-carbamoyl)glutathione (SMG) was inhibited when incubations were conducted in an atmosphere of CO:air (1:1) or when SKF 525-A (3.0mM) was included in the incubations. Pre-treatment of mice with phenobarbitone (PB, 80mg/kg for 4 days) or beta-naphthoflavone (BNF, 50mg/kg for 4 days) failed to increase the microsomal formation of SMG from NMF. This result suggests that the oxidation of NMF is catalysed by a cytochrome P-450 isozyme which is unaffected by PB or BNF. Microsomal incubations with DMF (5 or 10mM) failed to generate measurable amounts of SMG although DMF was metabolised to HMMF. Incubations of microsomes with HMMF resulted in the generation of a small amount of SMG which was affected by inhibitors of microsomal enzymes in the same way as in the case of NMF. HMMF was metabolised to AMCC by rodents in vivo. This result suggests that HMMF is a major intermediate in the metabolic activation of DMF.

The Role of protein kinase C modulation in the antiproliferative effects of bistratene A, bryostatin 1 and phorbol esters

PKC-mediated signalling pathways are important in cell growth and differentiation, and aberrations in these pathways are implicated in tumourigenesis. The objective of this project was to clarify the link between cell growth inhibition and PKC modulation.The PKC activators bryostatin 1 and 12-0-tetradecanoylphorbol-13-acetate (TPA) inhibited growth in A549 and MCF-7 adenocarcinoma cells with great potency, and induced HL-60 leukaemia cell differentiation. Bistratene A affected these cells similarly. Experiments were conducted to test the hypotheses that bistratene A exerts its effects via PKC modulation and that characteristics of cytostasis induced by bryostatin 1 and TPA depend upon PKC isozyme-specific events. After incubation of A549 cells with TPA or bistratene A, 2D phosphoprotein electrophoretograrns revealed three proteins phosphorylated by both agents. However, bistratene A was unable to induce the formation of cellular networks on the basement membrane substitute Matrigel, and staurosporine was unable to reverse bistratene A-induced [3H]thymidine uptake inhibition, unlike TPA. Bistratene A did not induce PKC translocation or downregulation, activate or inhibit A549 and MCF-7 cell cytosolic PKC or compete for phorbol ester receptors. Western blot analysis and hydroxylapatite chromatography identified PKC α, ε and ζ in these cells. Bistratene A was unable to activate any of these isoforms. Therefore the agent does not exert its antiproliferative effects by modulation of PKC activity. The abilities of bryostatin 1 and TPA (10nM-1μM) to induce PKC isoform translocation and downregulation were compared with antiproliferative effects. Both agents induced dose-dependent downregulation and translocation of PKC α and ε to particulate and nuclear cell fractions. PKC ζ was translocated to the particulate fraction by both agents in MCF-7 cells. The similarity of PKC isoform redistribution by these agents did not explain their divergent effects on cell growth, and the role of nuclear translocation of PKC in cytostasis was not confirmed by these studies. Alternative factors governing the characteristics of growth inhibition induced by these agents are discussed.

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.