Chem. Res. Toxicol. 1989,2,359-366
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Articles Role of Cytochrome P-450 I I E l and Catalase in the Oxidation of Acetonitrile to Cyanide Dennis E. Feierman and Arthur I. Cederbaum* Department of Biochemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029 Received April 24, 1989
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. 8,263-2711 have shown that microsomes oxidize acetonitrile to cyanide. Treatment of rats with inducers of cytochrome P-450 IIEl such as pyrazole, 4-methylpyrazole, and ethanol resulted in a 4- to &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 V,, per milligram of microsomal protein and per nanomole of P-450 but did not affect the apparent k, for acetonitrile, whereas the 4-methylpyrazole treatment increased V,, 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 IIEl 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 IIEl 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 inhibitor 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-H202 did not oxidize acetonitrile to cyanide under conditions in which other peroxidatic substrates such as ethanol were oxidized. A reconstituted system containing P-450 IIEl 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 IIEl is especially effective as a catalyst for this step. The second step involves peroxidation of this metabolite by catalase-H202, 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 IIEl as well as by acting as a competitive substrate for the peroxidatic activity of catalase-H202.
Introduction Nitriles are widely used in the chemical industry and as drugs (1-4). Acetonitrile is an important solvent and chemical intermediate that appears to have relatively little toxicity although occasional human fatalities have been reported (5-8). The acute toxicity of saturated nitriles appears to be due to the production of cyanide, and the * Address correspondence to this author at the Department of Biochemistry, Box 1020, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029.
toxicity of nitriles resembles that found with cyanide intoxication (4,9-13). The initial step in acetonitrile metabolism to cyanide has been suggested to involve a mixed-function oxidation at the a-carbon to yield the cyanohydrin (CH3CN HOCH,CN), with the subsequent release of cyanide (9).The detailed studies by Freeman and Hayes (14-16)have characterized microsomal oxidation of acetonitrile to cyanide. Formaldehyde was not a product of this reaction (14),suggesting that the overall oxidation pathway may be more complex than the simple scheme above. On the basis of the above and certain unusual kinetics, the possibility of a two-step pathway for aceto-
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0893-228~/89/2702-0359$01.50/0 0 1989 American Chemical Society
360 Chem. Res. Toxicol., Vol. 2, No. 6, 1989 nitrile oxidation was suggested, with one s t e p reflecting t h e mixed-function oxidase activity of cytochrome P-450 (15, 16). Ethanol and 4-methylpyrazole have been found t o decrease the lethality of succinonitrile and the production of cyanide; a two-step route for metabolizing nitriles to cyanide was proposed, with one step suggested t o involve alcohol dehydrogenase (17). However, ethanol and 4methylpyrazole also interact with microsomes, and ethanol inhibits acetonitrile oxidation t o cyanide b y microsomes (16). P r e t r e a t m e n t of r a t s with acetone has been found to result in a n increase in microsomal oxidation of acetonitrile to cyanide (15). Acetone induces cytochromes P-450 IIEl (P-45Oj) a n d P-450b/e in r a t liver (18, 19). T h e possibility that acetonitrile oxidation to cyanide is elevated by inducers of P-45Oj is of important toxicological significance, especially in alcoholics. Ethanol induces the P-45Oj isozyme (20-23), a n d alcoholics consume certain nitriles as antidepressants and as antialcohol adversive agents, e.g., cyanamide (Temposil). Experiments were conducted t o evaluate whether acetonitrile is an effective substrate for oxidation by P-450j, and to determine whether the production of cyanide from acetonitrile involves additional enzymes or steps besides that catalyzed by cytochrome P-450.
Materials and Methods Male Sprague-Dawley rats weighing about 135-150 g were utilized in all experiments and treated as previously described (24). Rats were injected intraperitoneally with either saline, pyrazole (200 mg/kg body weight/day for 2 days), 4-methylpyrazole (200 mg/kg body weight/day for 3 days), or phenobarbital (80 mg/kg/day for 4 days). Another group of rats was injected intraperitoneally with a suspension of 3-methylcholanthrene in corn oil (25 mg/kg/day for 3 days) or an equivalent volume of corn oil. The animals were starved overnight and sacrificed 24 h after the last injection. Hepatic microsomes were prepared by differential centrifugation, washed with and suspended in 125 mM KCl, and stored at -70 OC. For experiments involving chronic ethanol feeding, rats were fed a liquid diet for 3 weeks in which ethanol provided 36% of total calories, proteins 18%, fat 35%, and carbohydrate 11% (25). Pair-fed controls consumed the same diet except that carbohydrate isocalorically replaced ethanol. Prior to the day of sacrifice, the rats received their respective diets ad libitum. The oxidation of acetonitrile to cyanide was determined in 25-mL Erlenmeyer flasks at 37 OC by utilizing a basic reaction system consisting of 100 mM potassium phosphate, pH 7.4, 10 mM MgC12,0.4 mM NADP+, 100 mM acetonitrile, and about 0.3 mg of microsomal protein in a final volume of 1.0 mL. As will be described below, catalase was also required and was routinely added to the reaction system a t a final concentration of 13 000 units/mL. Reactions were initiated by the addition of a mixture of glucose &phosphate (final concentration of 10 mM) plus 2 units of glucose-6-phosphate dehydrogenase and terminated (usually after 20 min) by the addition of the reaction mixture to Conway tubes containing 0.4 mL of 100% trichloroaceticacid. The cyanide was recovered by microdiffusion (26)and analyzed colorimetrically at 586 nm by using a pyridine-barbituric acid reagent (27).' Standard c w e s were carried out with known amounts of sodium cyanide, and experimental values are corrected for recovery of cyanide. All values are also corrected for zero-time controls in which TCA was added prior to the NADPH-generating system. In experiments that involved determining the effect of carbon monoxide or nitrogen, the following procedure was used. The flasks (with a gas volume of about 30 mL) were sealed with serum stoppers prior to the addition of the NADPH-generating system. Ten cubic centimeters of air was removed with a gas-tight syringe We thank Dr. Eileen P.Hayes, Department of Environmental and Community Medicine, UMDNJ-Rutgers Medical School, for helping us with this assay procedure, for many helpful discussions, and for a generous gift of barbituric acid.
Feierman a n d Cederbaum
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Figure 1. Time courae for the oxidation of acetonitrile to cyanide by microsomes from saline controls ( 0 )or from rats treated with or Cmethylpyrazole (A). Experiment A refers either pyrazole (0) to activity per milligram of protein, and experiment B refers to activity per nanomole of P-450. followed by the immediate addition of 10 cm3 of carbon monoxide or nitrogen with a second syringe. After a 2-3-min incubation period, reactions were initiated with the NADPH-generating system. The catalase-H202-dependentoxidation of ethanol or of acetonitrile was determined in a reaction system containing 50 m M
KPi,pH7.4,0.1mMEDTA,0.4mMxanthine,O.lunitofxanthine oxidase, 300 units of catalase, and either 50 mM ethanol or 100 mM acetonitrile in a final volume of 1.0 mL. Reactions were initiated by the addition of xanthine oxidase and terminated by the addition of 1 mL of 1 N HC1. Acetaldehyde was determined by a head-space gas chromatography procedure (28). The pyrazole-induced cytochrome P-450 isozyme (P-450 IIE1) was purified from liver microsomes isolated from rats treated with pyrazole with minor modifications of the method previously described (29). The modifications consisted of the addition of butylated hydroxytoluene to a final concentration of 0.1 mM to the initial sucrose-Tris-EDTA homogenization buffer and the inclusion of 2 mM pyrazole in the equilibration buffer used in the f i a l step involving hydroxyapatite chromatography. The fiial P-450 preparation had a specific content of about 12 nmol of P-450/mg of protein and displayed a DMSO binding spectrum, and one major band of apparent molecular weight of 52000 was produced on SDS gel electrophoresis. The P-450 was stored in 50 mM potassium phosphate buffer, pH 7.4, containing 20% v/v glycerol a t -70 O C . NADPH-cytochrome P-450 reductase was purified (30)to a final specific activity of 40 rmol of cytochrome c reduced/(min.mg of protein). The oxidation of acetonitrile to cyanide was assayed in a reaction system containing 100 mM potassium phosphate, pH 7.4,200 mM acetonitrile, 13 000 units of catalase, and the previously reconstituted system consisting of 0.1 nmol of P-450, 0.2 unit of NADPH-cytochrome P-450 reductase, and 30 clg of sonicated dilaurylphosphatidylcholinein 50 mM phosphate buffer, pH 7.4, in a final volume of 1.0 mL. Reactions were initiated by the addition of NADPH to a final concentration of 1.0 mM and terminated as described above for the microsomal studies. The content of cytochrome P-450 was determined by the method of Omura and Sato (31). All buffers and the water used to prepare solutions were passed through columns of Chelex 100 resin to remove metals. All values refer to the mean f standard error. The number of experiments is indicated in the table footnotes.
Results Microsomes isolated from saline control rats oxidized acetonitrile to cyanide in a reaction linear with time t o at least 30 min (Figure 1)a n d with microsomal protein (up t o at least 1.0 m g / m L , data n o t shown). Little or n o cyanide was detected in t h e absence of microsomes, acetonitrile, or t h e NADPH-generating system. Rates of
Chem. Res. Toxicol., Vol. 2, No. 6, 1989 361
Oxidation of Acetonitrile to Cyanide
treatment saline pyrazole 4-methylpyrazole phenobarbital corn oil 3-methylcholanthrene pair-fed control chronic ethanol
Table I. Oxidation of Acetonitrile to Cyanide by Microsomes' rate of cyanide production, nmol/min content of P-450, nmol/mg per mg per nmol of P-450 0.92 f 0.02 0.32 f 0.03 0.35 f 0.03 0.95 f 0.04 1.49 f 0.34 1.57 f 0.29 1.52 f 0.11 2.14 f 0.24 1.41 f 0.28 0.74 0.09 0.47 f 0.13 1.57 f 0.24 0.83 i 0.04 0.87 f 0.07 1.06 f 0.07 1.65 f 0.11 0.30 f 0.10 0.18 f 0.12 0.61 0.11 0.18 1.19 0.96 0.81
effect of treatmentb per mg per nmol of P-450 +366 +569 +131 (+172) -66 (-6)
+349 +303 +34 (+203) -83 (-49)
+773
+350
a The oxidation of 100 mM acetonitrile to cyanide by microsomes from rats treated with either pyrazole, 4-methylpyrazole, phenobarbital, 3-methylcholanthrene, or ethanol and their respective controls was assayed as described under Materials and Methods. Results are from four experiments except for the chronic ethanol and pair fed, where two pairs of rats were used. bThe numbers in parentheses refer to the effect of the corn oil or 3-methylcholanthrene treatment relative to the saline control.
cyanide production [about 0.3 nmol/ (mimmg of microsomal protein)] are similar to those reported by Freeman and Hayes (16). Microsomes isolated from rats treated with pyrazole or 4-methylpyrazole displayed an increased effectiveness in oxidizing acetonitrile to cyanide (Figure 1A). Previous results indicated that pyrazole treatment had little effect on the total content of cytochrome P-450, whereas 4-methylpyrazole increased the total P-450 content (32). When rates of cyanide production were expressed per nanomole of total cytochrome P-450, pyrazole and 4-methylpyrazole treatment increased the oxidation of acetonitrile 4- to 5-fold (Figure 1B). The increase in acetonitrile oxidation by microsomes after pyrazole treatment suggests that P-45Oj is an effective catalyst for oxidizing this substrate since pyrazole induces this isozyme of P-450 (29, 33, 34). The effects of other inducers of P-450 were also evaluated to assess their relative specificity for increasing oxidation of acetonitrile. Treatments included chronic ethanol consumption, which induces P-450j, and phenobarbital or 3-methylcholanthrene, which do not induce this isozyme. Time courses for acetonitrile oxidation to cyanide were linear for 30 min with all the microsomal preparations (data not shown). Since most of the above inducers increase the content of P-450, results in Table I are expressed per milligram of protein and per nanomole of P-450. Treatment with pyrazole, Cmethylpyrazole,and ethanol resulted in a 4- to 5-fold increase in acetonitrile oxidation per nanomole of P-450, whereas phenobarbital treatment (per nanomole of P-450) had only a small stimulatory effect; 3-methylcholanthrene treatment decreased acetonitrile oxidation relative to the corn oil control or even the saline control. It is of interest that the corn oil treatment itself increased acetonitrile oxidation (Table I); we have previously noted that corn oil also increased microsomal oxidation of pyrazole and binding of pyrazole and 4methylpyrazole to microsomes, suggesting the possible induction of P-45Oj by this treatment itself (24,35). The increased effectiveness of microsomes from pyrazole-, 4methylpyrazole-, and ethanol-fed rats, as compared to phenobarbital or 3-methylcholanthrene treatment, in oxidizing acetonitrile to cyanide suggests that acetonitrile is an effective substrate for metabolism by P-45Oj. To evaluate whether the pyrazole or 4-methylpyrazole treatment affects the affinity for acetonitrile, the oxidation of varying concentrations of acetonitrile by microsomes was determined. Results in Figure 2 show that cyanide formation was increased as the concentration of acetonitrile was elevated from 10 to 100 mM with all the microsomal preparations. At all concentrations of acetonitrile utilized, cyanide production was highest with the microsomes from the rats treated with pyrazole and 4-methylpyrazole,
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Figure 2. Substrate concentration curve for the oxidation of acetonitrile to cyanide by microsomes. The concentration of acetonitrile was varied from 10 to 100 mM. Experiment A refers to nanomoles of cyanide produced per minute per milligram of microsomal protein, while experiment B refers to activity per nanomole of cytochrome P-450. Results are from three or four experiments. The following microsomal preparations were utilized: (0) saline control; (0) pyrazole; (A)4-methylpyrazole; ( 0 )phenobarbital; (w)corn-oil; (A)3-methylcholanthrene.
whether results were expressed per milligram of protein or per nanomole of P-450. The increase produced by the corn oil treatment, the slight stimulatory effect of phenobarbital, and the poor effectiveness of 3-methy:-
362 Chem. Res. Toxicol., Vol. 2, No. 6, 1989
Feierman and Cederbaum
Table 11. Kinetic Parameters for the Oxidation of Acetonitrile to Cyanide by Microsomes" V-, per mg V, per nmol V-(per nmol microsomes k,, mM of protein of P-450 of P-450)/km saline 28 f 7 0.33 f 0.06 0.36 f 0.06 0.013 21 f 8 1.55 f 0.36 1.68 f 0.33 0.080 pyrazole 4-methylpyrazole 13 f 2 3.45 f 0.61 2.23 f 0.71 0.172 14 f 3 0.81 f 0.16 0.59 f 0.10 0.042 phenobarbital 30 f 6 0.98 f 0.17 1.14 f 0.34 0.038 corn oil 3-methylcholanthrene 26 f 1 0.34 f 0.11 0.20 f 0.06 0.008
"Kinetic parameters were calculated by regression analysis of Hanes-Woolf plots of the substrate concentration curves shown in Figure 2. Results are from three to four experiments. V, refers to nmol of cyanide produced/min. Table 111. Effect of Carbon Monoxide on Microsomal Oxidation of Acetonitrile to Cyanide' rate of cyanide formation, nmol/(min.mg of protein) minus plus gas mixture xanthine oxidase xanthine oxidase 100% air 0.82 0.83 67% air-33% N2 0.77 0.84 67% air-33% CO 0.12 0.08
" The oxidation of acetonitrile to cyanide by microsomes from pyrazole-treated rata was assayed as described under Materials and Methods under the indicated gas mixtures. In some experiments, a source of HzO, (0.4 mM xanthine plus 0.1 unit of xanthine oxidase) was added prior to the gassing of the flasks. Results are from two experiments. cholanthrene treatment in induction of acetonitrile oxidation were observed at all the concentrations of acetonitrile evaluated (Figure 2). Lineweaver-Burk and Hanes-Woolf plots of these data were linear (data not shown), and kinetic parameters were determined by regression analysis of the Hanes-Woolf plots (correlation coefficients >0.95). Pyrazole treatment had no effect on the affinity by microsomes for acetonitrile but increased the apparent V,, for acetonitrile oxidation (Table 11). The corn oil treatment also increased V,, without any effect on the apparent k, for acetonitrile whereas the 3-methylcholanthrene treatment lowered V,, per nanomole of P-450. Phenobarbital treatment lowered the apparent k, and slightly increased V,, (per nanomole of P-450) for acetonitrile oxidation (Table 11). Treatment with 4-methylpyrazole resulted in an increased affinity for acetonitrile as well as an increase in the apparent Vmm. The "catalytic efficiency" of the microsomes for oxidizing acetonitrile to cyanide was calculated by dividing the respective v,, values per nanomole of P-450 by the k, value for acetonitrile. These results are shown in the last column of Table 11. Pyrazole and 4-methylpyrazole treatment increased the catalytic efficiency by about 6- and 13-fold, respectively; the greater effect of 4-methylpyrazole probably reflects the elevation in V,, coupled to the lowering of the k,. Phenobarbital treatment increased the efficiency by about 3-fold, largely due to the lowering of the k, for acetonitrile. The 3-methylcholanthrene treatment did not enhance the catalytic efficiency of the microsomes for oxidizing acetonitrile. To implicate a role for cytochrome P-450 in the oxidation of acetonitrile, the effect of carbon monoxide and competitive drug substrates was determined. Replacing air with a mixture containing 33% carbon monoxide resulted in striking inhibition of acetonitrile oxidation (Table 111). A comparable concentration of nitrogen had no effect on cyanide formation, indicating that the inhibition by carbon monoxide was not due to a partial anaerobic effect. Effective substrates for P-45Oj include ethanol and 2-butanol (20-23, 36,37);DMSO has been shown to interact with P-45Oj and produce a substrate binding spectrum (38);
Table IV. Effect of Substrates on Microsomal Oxidation of Acetonitrile to Cyanide" rate of cyanide formation, addition concn, mM nmol/(min.mg) effect, % control 1.75 0.44 -75 ethanol 1 0.16 5 -90 2-butanol 1 0.49 -7 2 5 0.08 -95 4-methylpyrazole 0.2 0.14 -92 DMSO 5 0.65 -63 50 -95 0.09
" The oxidation of acetonitrile to cyanide by microsomes from pyrazole-treated rata was assayed in the absence or presence of the indicated substrates. Results are from two experiments.
0
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Figure 3. Effect of anti-P-450 3a IgG (0, 0)or normal sheep serum IgG (0,W) on the oxidation of acetonitrile by microsomes m) from pyrazole-treated (O,.) or 4-methylpyrazole-treated (0, rats. Microsomes were incubated with the indicated amounts of IgG for 5 min prior to the addition of acetonitrile and the NADPH-generating system.
4-methylpyrazole has also been shown to bind effectively to P-45Oj and to be a potent inhibitor of the P-450j-catalyzed oxidation of ethanol (35,39). All these compounds produced striking inhibition of the oxidation of acetonitrile by microsomes from pyrazole-treated rats (Table IV). Control and recovery experiments indicated that these compounds did not interfere with the assay or recovery of standard amounts of cyanide. Antibody specific against the P-450 3a isozyme of rabbit liver microsomes was shown to also recognize P-450 IIEl of rat liver microsomes and to inhibit the oxidation of aniline and 1-butanol by microsomes isolated from rats treated with inducers of P-450 IIEl (23). We recently found that the anti-P-450 3a IgG at concentrations of 2-4 mg of IgG/nmol of total P-450 produced about 70% inhibition of the oxidation of pyrazole to 4-hydroxypyrazole by microsomes isolated from rats treated with pyrazole and 4-methylpyrazole (40). As shown in Figure 3, increasing amounts of the anti-P-450 3a IgG produced progressive inhibition of the production of cyanide from acetonitrile by microsomes from pyrazole-treated rats. At concentrations of 2-4 mg of IgG/nmol of P-450, the oxidation of
Chem. Res. Toxicol., Vol. 2, No. 6, 1989 363
Oxidation of Acetonitrile to Cyanide Table V. Effect of Azide on Cyanide Production from Acetonitrile Experiment A" rate of cyanide formation, nmol/(min.mg of protein) microsomes -azide +azide 0.31 f 0.04
