Metabolic oxidation and toxification of N-methylformamide catalyzed by the cytochrome P450 isoenzyme CYP2E1.
Alkylformamides, for example N-methylformamide, are hepatotoxic in rodents and humans. The mechanism by which N-methylformamide exerts its hepatotoxicity involves metabolic oxidation at the formyl moiety to yield a short-lived intermediate, perhaps methyl isocyanate, which reacts with glutathione to afford S-(N-methylcarbamoyl)glutathione. The hypothesis that the cytochrome P450 isozyme CYP2E1 catalyzes the metabolic toxification of N-methylformamide was tested. Hepatocytes obtained from mice that had received acetone, an inducer of CYP2E1, were incubated for up to 4 hr with N-methylformamide (5 and 10 mM). Whereas N-methylformamide caused cytotoxicity in these cells, as measured by release from the cells of lactate dehydrogenase, it was barely toxic, under these conditions, to cells from untreated mice. Coincubation of N-methylformamide with dimethylsulfoxide (10 mM), a CYP2E1 inhibitor, for 4 or 6 hr abolished the hepatocytotoxicity of N-methylformamide. Metabolism of N-methylformamide to S-(N-methylcarbamoyl) glutathione was measured in incubates with liver microsomes from rats, mice, or humans in the presence of glutathione. Pretreatment of rodents with acetone or ethanol induced the rate of metabolism of N-methylformamide and of p-nitrophenol, a known CYP2E1 substrate, but it did not increase aminopyrine N-demethylation. Metabolism of N-methylformamide and p-nitrophenol was elevated in microsomes from animals that had received acetone (1%) in their drinking water for 1 week to 230% and 200%, respectively, of control values in mouse microsomes and to 310% and 240%, respectively, of control values in rat microsomes. Pretreatment of animals with 4-methylpyrazole (200 mg/kg intraperitoneally, once daily for 3 days) increased metabolism of N-methylformamide to 410% of control values in rat liver microsomes but was without effect on murine microsomal metabolism of N-methylformamide. The metabolism of this compound was strongly inhibited by the CYP2E1 substrates or inhibitors dimethylsulfoxide (1-100 mM), p-nitrophenol (100 microM), and diethyldithiocarbamate (100 microM), which did not affect aminopyrine N-demethylation. A polyclonal antibody against rat CYP2E1 (10 mg of IgG/nmol of cytochrome P450) inhibited N-methylformamide metabolism in liver microsomes from rats and from a human by 75% and 80%, respectively. The rate of metabolism of N-methylformamide to S-(N-methylcarbamoyl) glutathione was determined in liver microsomes from six humans and correlated with extent of metabolic hydroxylation of chlorzoxazone, a CYP2E1 probe, and with amount of immunodetectable enzyme using an anti-rat CYP2E1 antibody (r = 0.81 and 0.80, respectively). The results suggest that CYP2E1 is the predominant, if not sole, cytochrome P450 isozyme responsible for the metabolic toxification of hepatotoxic N-alkylformamides.
Hyland
R
R
Pharmaceutical Sciences Institute, Aston University, Birmingham, UK.
Gescher
A
A
Thummel
K
K
Schiller
C
C
Jheeta
P
P
Mynett
K
K
Smith
A W
AW
Mráz
J
J
D000096:Acetone / Q000494:pharmacology; D000818:Animals; D001711:Biotransformation; D002478:Cells, Cultured; D002753:Chlorzoxazone / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004790:Enzyme Induction; D005260:Female; D005559:Formamides / Q000378:metabolism / Q000493:pharmacokinetics* / Q000633:toxicity; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains
D065607:Cytochrome P-450 Enzyme Inhibitors; D005559:Formamides; D007527:Isoenzymes; D000096:Acetone; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D002753:Chlorzoxazone; C002950:methylformamide; D004121:Dimethyl Sulfoxide