Role of cytochrome P 450 IIE1 and catalase in the oxidation of acetonitrile to cyanide

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Abstract
Acetonitrile is a common industrial solvent and laboratory agent, which can be toxic if ingested. The toxicity of nitriles appears to be due to the production of cyanide, and detailed studies by Freeman and Hayes [(1988) Biochem. Pharmacol. 37, 1153-1159; (1987) Fundam. Appl. Toxicol. 8, 263-271] have shown that microsomes oxidize acetonitrile to cyanide. Treatment of rats with inducers of cytochrome P-450 IIE1 such as pyrazole, 4-methylpyrazole, and ethanol resulted in a 4- to 5-fold increase in cyanide production from acetonitrile by isolated microsomes. Phenobarbital treatment had a small stimulatory effect, whereas 3-methylcholanthrene treatment decreased microsomal oxidation of acetonitrile. Pyrazole treatment increased Vmax per milligram of microsomal protein and per nanomole of P-450 but did not affect the apparent km for acetonitrile, whereas the 4-methylpyrazole treatment increased Vmax and the apparent affinity for acetonitrile. Cyanide production was inhibited by carbon monoxide as well as by substrates and compounds that interact with the P-450 IIE1 isozyme such as ethanol, 2-butanol, DMSO, and 4-methylpyrazole. Oxidation of acetonitrile to cyanide by microsomes from rats treated with pyrazole or 4-methylpyrazole was nearly completely inhibited by anti-P-450 3a IgG. These results implicate a role for P-450 in the oxidation of acetonitrile to cyanide and suggest that P-450 IIE1 may be an especially effective catalyst for this oxidation. Acetonitrile oxidation was not affected by hydroxyl radical scavengers or by desferrioxamine, indicating no role for hydroxyl radicals in the overall mechanism. Azide, an inhibitior of catalase, completely blocked cyanide production from acetonitrile by all microsomal preparations; added catalase overcame the azide inhibition. Formate, a substrate for the peroxidatic activity of catalase, also blocked microsomal oxidation of acetonitrile. These results implicate a role for catalase in the microsomal oxidation of acetonitrile; however, catalase-H2O2 did not oxidize acetonitrile to cyanide under conditions in which other peroxidatic substrates such as ethanol were oxidized. A reconstituted system containing P-450 IIE1 purified from pyrazole-treated rats oxidized acetonitrile to cyanide; the presence of catalase was required for this production of cyanide. These results suggest that the overall oxidation of acetonitrile to cyanide is mediated by a two-step process. The first step involves oxidation of acetonitrile by cytochrome P-450 to a hydroxylated metabolite. P-450 IIE1 is especially effective as a catalyst for this step. The second step involves peroxidation of this metabolite by catalase-H2O2, with the subsequent release of cyanide. Ethanol, which has been found useful in preventing toxicity of nitriles, may act by competing with acetonitrile for oxidation by P-450 IIE1 as well as by acting as a competitive substrate for the peroxidative activity of catalase-H2O2.