Introduction

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Acrylonitrile (AN²) is widely used in the production of acrylic and modacrylic fibers, plastics, rubbers, resins, and as a chemical intermediate in the synthesis of many other industrial products (IARC, 1999). Early epidemiological studies have suggested that AN may increase the incidence of lung, colon, and stomach cancers among exposed workers (Thiess and Fleig, 1978; Blair et al., 1998). However, recent studies suggested that there is no significant association between human exposure to AN and carcinogenicity (Benn and Osborne 1998; Swaen et al., 1998; Wood et al., 1998; IARC, 1999; Marsh et al., 2001; Schulz et al., 2001). Animal studies demonstrated that AN is mutagenic, teratogenic, and a multisite carcinogen in rats and mice (Strother et al., 1988; IARC, 1999; Saillenfait and Sabate, 2000; NTP, 2001; Ghanayem et al., 2002).

AN metabolism and disposition are well characterized. It is directly conjugated with glutathione (GSH) and epoxidized by cytochrome P450 enzymes (P450) to form cyanoethylene oxide (CEO) (Fig. 1). It was hypothesized that CEO hydrolysis via epoxide hydrolase (EH) is the primary route of cyanide formation (Fig. 1). Alternatively, CEO may undergo rearrangement followed by hydride transfer to yield cyanide and acetic acid. Cyanide is converted to thiocyanate via rhodanese and eliminated in the urine. Both AN and CEO react with tissue thiols, leading to rapid depletion of GSH (Farooqui and Ahmed, 1983; Ghanayem et al., 1985; Benz et al., 1997; Nerland et al., 2001). CEO was shown to react with DNA and is mutagenic, which suggested that CEO may be responsible for the carcinogenic effects of AN (Guengerich et al., 1981).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   A proposed scheme showing the role of cytochrome P450 and epoxide hydrolase enzymes in the metabolism of acrylonitrile to cyanide. GSH, glutathione

Although the metabolic basis of the acute toxicity of AN has not been fully elucidated, it is generally attributed to its metabolism to CEO and cyanide, and glutathione depletion. The primary target of acute toxicity of AN is the central nervous system due, at least partially, to the liberation of cyanide (Ahmed and Patel, 1981; Benz et al., 1997). Cyanide-like symptoms are observed in animals after administration of AN (Graham, 1965; Ahmed and Patel, 1981), and cyanide antidotal regimens have been used in the United States to counter the acute toxicity of this chemical in humans (Benz et al., 1997). High doses of N-acetylcysteine are recommended in Germany for the treatment of acute AN poisoning in humans (Buchter et al., 1984), and sulfur-containing compounds were used to suppress AN metabolism and toxicity in animals (Benz et al., 1990). AN administration to rats also causes acetylcholine-like toxicity, an effect that was attributed to modulation of tissue sulfhydryls (Ghanayem et al., 1985, 1991).

A study by Abreu and Ahmed (1980) demonstrated that in vitro conversion of AN to cyanide is a P450-dependent pathway. It was suggested that whereas CYP2E1 plays a major role in the metabolism of AN to CEO, other cytochrome P450s (P450s) are also involved (Guengerich et al., 1991; Kedderis et al., 1993; Subramanian and Ahmed, 1995). Recent investigation showed that metabolism of AN was markedly increased in hepatic and extrahepatic rat microsomes obtained from animals treated with P450s inducers. This further suggested that multiple P450s may be involved in the metabolism of AN to cyanide (Mostafa et al., 1999). Earlier studies showed that inducers (phenobarbital) and inhibitors (SKF 525A) of P450 enzymes had no effect on thiocyanate excretion in animals (Gut et al., 1975). In contrast, recent studies showed that CEO-derived mercapturic acids, which are normally identified in the urine of AN-treated mice, were not detectable in the urine of CYP2E1-null mice treated with this chemical (Sumner et al., 1999; Ghanayem et al., 2000). It was therefore concluded that CYP2E1 is the only enzyme responsible for the epoxidation of AN to CEO in mice (Sumner et al., 1999; Ghanayem et al., 2000). Current studies were designed to more directly assess the role of CYP2E1 in the metabolism of AN to cyanide using CYP2E1-null versus wild-type mice. Furthermore, since EH is considered essential in the metabolism of CEO to cyanide, EH expression in CYP2E1-null and wild-type mice was also compared.

	Materials and Methods

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Chemicals. Acrylonitrile (99% pure, containing 1% hydroquinone as a stabilizer) was purchased from Sigma-Aldrich (St. Louis, MO). Pyridine-barbituric acid reagent, chloramine-T trihydrate, and potassium cyanide were purchased from Fisher Scientific (Pittsburgh, PA). CYP2E1 Western blotting kit and nitrocellulose membranes were obtained from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Purified recombinant human CYP2E1 protein was purchased from Research Diagnostics Inc. (Flanders, NJ). Antibodies to detect soluble and microsomal epoxide hydrolases, as well as purified rat protein standards, were gifts from Dr. Bruce Hammock (University of California at Davis). All other chemicals were of reagent grade purity.

Animals and Treatments. Male wild-type (CYP2E1+/+) and CYP2E1-null (CYP2E1/, CYP2E1-null) mice were obtained from a colony developed at the laboratories of Dr. Frank Gonzalez, National Cancer Institute, Bethesda, Maryland (Lee et al., 1996). The background of these animals is 129/Sv. Mice were rederived and bred at Charles River Laboratories, Inc. (Wilmington, MA). Weighing 25 to 30 g (4-6 months old), animals were quarantined for 1 week before use in temperature and humidity controlled rooms with a 12-h light/dark cycle. National Institutes of Health 31 rodent chow and tap water were provided ad libitum. All animal care and procedures were according to the National Institutes of Health guidelines (NIH, 1986). Dosing solutions were prepared in tap water in a dose volume of 10 ml/kg body weight and were immediately used to avoid potential polymerization of AN. A single gavage dose of AN was administered at 0 (vehicle control), 2.5, 10, 20, or 40 mg/kg.

Tissue Collection. One or three hour(s) after AN administration, animals were euthanized using CO₂/O₂ mixture. Blood was collected in a heparinized syringe by cardiac puncture. Approximately 1 ml of whole blood was immediately mixed with 20 µl of 0.125 M sodium nitrite and frozen in liquid N ₂ (Benz et al., 1997). Liver, kidneys, brain, and lungs were excised, rinsed in ice-cold phosphate-buffered saline (pH 7.4), and frozen in liquid N₂. All blood and tissues were then stored at 80°C until analysis.

Cyanide Measurements. Cyanide concentrations in blood and tissue homogenates were determined by a modified spectrophotometric method (Baselt, 1988; Benz et al., 1997). Specimens (200-300 mg) of kidney, lung, or liver were homogenized in cold 0.1 M sodium phosphate buffer (pH 7.4). Brain tissue was processed as a 20% homogenate in 20 mM silver sulfate (Lundquist et al., 1985). One milliliter of 0.1 M NaOH was added to the central well of a Conway diffusion cell. Three milliliters of 15% sulfuric acid was added to one side of the middle well, and nitrated blood (1 ml) or tissue homogenate was added to the opposite side of the middle well. The Conway dish was then sealed using silicone grease and left to incubate on a horizontal shaker at 70 rpm for 3 h at room temperature. At the end of incubation, 0.5 ml of NaOH from the central cell of the dish was added to a cuvette containing 0.2 ml of 1 M potassium phosphate buffer (pH 3.9). Fifty microliters of 0.25% chloramine-T were added to the cuvette and allowed to react at room temperature for 3 min. Five hundred microliters of pyridine-barbituric acid reagent were added to the cuvette, and the maximum absorbance at 585 nm was determined by monitoring over 5 min. Serially diluted standards of potassium cyanide in 0.1 M NaOH were used to construct a standard curve. The limit of cyanide detection of this method was found to be less than 1.0 nmol CN/ml.

Microsomal Preparation and Western Blot Analysis. Liver, lung, and kidney from male wild-type and CYP2E1-null mice were homogenized at 4°C in buffer (25% w/v) containing 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), and 0.25 mM phenylmethylsulfonyl fluoride. The homogenates were centrifuged at 10,000g for 20 min at 4°C. The supernatant was transferred to a new tube and centrifuged at 100,000g for 70 min at 4°C. After centrifugation, the supernatant was retained as the cytosolic fraction, and the microsomal pellet was resuspended in buffer [50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 1 mM EDTA, and 20% glycerol]. Protein concentrations were determined using the bicinchoninic acid method (Pierce Chemical, Rockford, IL). Ten micrograms of microsomal or cytosolic protein were electrophoretically separated on a 4 to 12% gradient Bis-Tris gel and transferred to a 0.45-µm nitrocellulose membrane. Immunoreactive CYP2E1 protein present in different samples was detected using a rat CYP2E1 antibody. Purified recombinant human CYP2E1 protein was used as a running standard.

Statistical Analysis. Statistical significance (control versus treatment groups) was determined using Student's t test. All results are presented as mean ± S.E. and P  0.05 are considered statistically significant.

	Results

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Comparison of the Acute Toxicity of AN in CYP2E1-Null and Wild-Type Mice. Cyanide-like toxicity was observed in AN-treated male wild-type mice in a dose-dependent manner. Within minutes after AN administration, animals started to exhibit irregular breathing, trembling, and convulsions. The severity of these symptoms increased as a function of dose, reaching maximum around 1 h after dosing with 10 or 20 mg/kg, and animals showed signs of recovery at the 3-h time point. In the 40 mg/kg dose group, the toxicity of AN was more severe and all male wild-type mice died before the 3 h time point. Male CYP2E1-null mice showed no symptoms of intoxication at any time during the study and no deaths occurred among these mice at doses as high as 40 mg/kg.

Cyanide Concentrations in Blood and Tissues of CYP2E1-Null and Wild-Type Mice. Cyanide concentrations in blood and tissues of male mice are shown in Figs. 2 and 3. A dose-dependent increase in blood cyanide concentrations was found 1 and 3 h after AN administration in male wild-type mice (Fig. 2). Compared with tissues of wild-type mice sampled in this study, blood had the highest concentration of cyanide. Blood cyanide concentrations in male wild-type mice peaked 1 h after AN administration regardless of dose and correlated well with toxicity, suggesting an association between cyanide levels and the observed acute toxicity of this chemical. At 3 h after AN administration, blood cyanide levels declined and were lower than the levels observed at 1 h. At 40 mg of AN/kg, all male wild-type mice died before the 3-h observation time point. In contrast, male CYP2E1-null mice treated with AN at doses as high as 40 mg/kg showed no significant increase (compared with vehicle-treated controls) in the concentration of cyanide in blood (Fig. 2).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effect of dose and time on the concentration of cyanide in blood of male CYP2E1-null and wild-type mice after gavage administration of acrylonitrile. Values are presented as the mean ± S.E. of three to four animals. *, indicates cyanide values in wild-type mice, which are statistically different from similarly treated CYP2E1-null mice at p  0.05. Male wild-type mice, 1 h (); male wild-type mice, 3 h (); CYP2E1-null mice, 1 h (); CYP2E1-null mice, 3 h ().

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of dose and time on the concentration of cyanide in liver, lung, kidney, and brain tissues of male CYP2E1-null and wild-type mice after gavage administration of acrylonitrile. Values are presented as the mean ± S.E. of three to four animals. *, indicates cyanide values of wild-type mice which are statistically different from similarly treated CYP2E1-null mice at p  0.05. Male wild-type mice, 1 h (); male wild-type mice, 3 h (); CYP2E1-null mice, 1 h (); CYP2E1-null mice, 3 h ().

Expression of CYP2E1 and EH in CYP2E1-Null and Wild-Type Mice. Immunoblotting results revealed that CYP2E1 is well expressed in the liver, kidney, and lung of male wild-type mice, and as expected, no expression of this enzyme was detected in the tissues of CYP2E 1-null mice (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of immunoreactive mEH and sEH in liver of male wild-type and CYP2E1-null mice. Each lane (except where noted) contains 10 µg of microsomal or cytosolic protein. A, mEH protein content, lanes 1 to 3 represent three wild-type mice, lanes 4 to 6 represent three CYP2E1-null mice, and lane 4 is standard (S) of 50 ng of purified rat mEH. B, sEH protein content, lanes 1 to 3 represent three wild-type mice, lanes 4 to 6 represent three CYP2E1-null mice, and lane 7 (S) is 80 ng of purified rat sEH standard. C, mean relative density ± 1 S.E. of protein bands from Western blots. Closed bars indicate wild-type samples; open bars indicate CYP2E1-null samples.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Western blot analysis of immunoreactive mEH and sEH protein in kidneys of male wild-type and CYP2E1-null mice. Each lane (except where noted) contains 10 µg of microsomal or cytosolic protein. A, mEH protein content, lanes 1 to 3 represent three wild-type mice, lanes 5 to 7 represent three CYP2E1-null mice, and lane 4 (S) represents 50 ng of purified rat mEH standard. B, sEH protein content, lanes 2 to 4 represent three wild-type mice, lanes 5 to 7 represent three CYP2E1-null mice, and lane 1 (S) represent 80 ng of purified rat sEH standard. C, mean relative density ± 1 S.E. of protein bands from Western blots. Closed bars indicate wild-type samples; open bars indicate CYP2E1-null samples. S, standard.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Western blot analysis of immunoreactive microsomal and soluble epoxide hydrolase protein in lungs of male wild-type and CYP2E1-null mice. Each lane (except where noted) contains 25 µg of microsomal or cytosolic protein. A, mEH protein content, lanes 2 to 4 represent three wild-type mice, lanes 5 to 7 represent three CYP2E1-null mice, and lane 1 (S) represents 50 ng of purified rat mEH standard. B, sEH protein content, lanes 1 to 3 represent 3 wild-type mice, lanes 4 to 6 represent 3 CYP2E1-null mice, and lane 7 (S) represents 80 ng of purified rat sEH standard. C, mean relative density ± 1 S.E. of protein bands from the Western blots. Closed bars indicate wild-type samples; open bars indicate CYP2E1-null samples. S, standard.

	Discussion

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Acrylonitrile is a potent acute toxicant and known animal carcinogen. It is believed that metabolism is a prerequisite for the development of the acute and chronic toxicities of AN. A number of studies have demonstrated that CYP2E1 plays an important role in the epoxidation of AN; however, it was also reported that other P450s are involved (Guengerich et al., 1991; Kedderris et al., 1993; Subramanian and Ahmed, 1995). It was hypothesized that AN metabolism to CEO via CYP2E1 precedes cyanide formation and that EH plays an essential role in the release of cyanide from CEO (Fig. 1). Present investigations were undertaken to examine this hypothesis. Using ¹³C NMR, urinary metabolites identification in mice treated with ¹³C-AN demonstrated that whereas CEO-derived urinary metabolites accounted for more than 80% of the dose in wild-type mice, they were not detectable in the urine of CYP2E1-null mice (Sumner et al., 1999; Ghanayem et al., 2000). It was concluded from this work that AN oxidation to CEO is exclusively catalyzed by CYP2E1. Current work is a follow-up study designed to more directly assess the role of CYP2E1 in the metabolism of AN to cyanide by comparing cyanide concentrations in CYP2E1-null versus wild-type mice. Additionally, this work included a comparison of the expression of CYP2E1 and EH in mice of both genotypes.

Results presented here indicated that after AN administration to wild-type mice, cyanide levels increased in blood and tissues in a dose-dependent manner. Cyanide levels were higher in blood than in other tissues at all time points and at all doses of AN up to 40 mg/kg. As the major AN-metabolizing organ (Ahmed et al., 1996), liver contained higher cyanide levels than other organs. These findings support the hypothesis that AN metabolism to cyanide occurs primarily in the liver and is transported by the blood to other tissue. Data presented in the current study also demonstrated that cyanide concentrations in blood and tissues of CYP2E1-null mice administered AN were not statistically different from those measured in vehicle-treated animals. Furthermore, no symptoms of toxicity were observed at any doses of AN given to CYP2E1-null mice. Whereas all wild-type male mice that received 40 mg of AN/kg died less than 3 h after chemical administration, CYP2E1-null mice survived. Subsequent studies in this laboratory demonstrated that CYP2E1-null mice could survive doses of AN as high as 60 mg/kg/day, 5 days/week for 6 weeks (data not shown). These results showed that absence of CYP2E1 prevented the metabolism of AN to cyanide and protected mice from the acute toxicity and lethality of this chemical. This data supports the hypothesis that oxidative metabolism is essential for the metabolism of AN to cyanide. Contrary to earlier reports, which suggested that glutathione depletion is a critical event in the toxicity of AN, current work showed that AN metabolism to CEO and its subsequent metabolism to cyanide are critical events in the development of the acute toxicity/mortality of AN in mice. Prevention of CEO (Sumner et al., 1999; Ghanayem et al., 2000) and cyanide formation and the absence of observable acute toxicity in CYP2E1-null mice at high doses of AN further suggest that inhibition of CYP2E1 may be an efficient therapeutic intervention in the treatment of AN intoxication.

Earlier studies (Sumner et al., 1999) demonstrated that no urinary metabolites derived from CEO are found in the urine of CYP2E1-null mice treated with AN and clearly confirmed that CYP2E1 is the only P450 enzyme responsible for AN epoxidation. This finding in conjunction with current data demonstrated that CYP2E1-mediated epoxidation of AN to CEO is a prerequisite for cyanide formation in mice. This conclusion was further supported by the fact that inhibition of P450s by ABT prior to AN administration to wild-type mice led to negligible cyanide formation and toxicity in these animals. Current results further demonstrated that no P450s other than CYP2E1 are involved in the metabolism of AN to cyanide in mice.

Results from in vitro investigations using chemical modulators of P450s indicated that activation of AN to cyanide was markedly enhanced in microsomes obtained from rats treated with known P450 inducers like phenobarbital, ethanol, and 3-methylcholanthrene (Mostafa et al., 1999). It was concluded that these modulators of P450s were responsible for the increased metabolism of AN to cyanide. Elevated in vivo blood cyanide levels were reported in rats treated for 7 days with phenobarbital followed by AN (Ahmed and Patel, 1981). However, recent studies by Gerhold et al. (2001) showed that phenobarbital strongly induces mEH (but not sEH) in rat liver. In light of the present results, it is likely that increased cyanide formation by phenobarbital-induced microsomes or in phenobarbital-treated animals may, at least partially, be due to increased EH expression. Earlier in vitro studies of AN metabolism showed that cyanide formation was inhibited with the addition of 1,1,1-trichloropropane 2,3-oxide, a potent EH inhibitor (Abreu and Ahmed, 1980). We therefore compared the expression of EH in mice of both genotypes to account for the possibility that CEO may be formed in CYP2E1-null mice via non-CYP2E1 enzymes and subsequently release cyanide via EH. Although we detected a slight increase in the expression of sEH in the liver of wild-type versus CYP2E1-null mice, it is believed that this minor difference in the expression of sEH is insufficient to explain the observed major differences in AN metabolism to cyanide in mice of both genotypes. This further confirmed that CYP2E1-null mice possess all the enzymatic machinery (except CYP2E1) necessary to metabolize AN to cyanide and that the absence of cyanide formation in these animals is exclusively caused by the deletion of CYP2E1.

In conclusion, under the current experimental conditions using CYP2E1-null mice, present studies demonstrated for the first time that CYP2E1-mediated oxidation of AN is a prerequisite for AN metabolism to cyanide. This work also suggested that no other pathways or enzymes are involved in the initial step of AN metabolism that subsequently leads to cyanide formation. Earlier studies also demonstrated that CYP2E1 is the only enzyme responsible for AN epoxidation to CEO in mice (Sumner et al., 1999; Ghanayem et al., 2000). These data collectively demonstrated that AN metabolism to CEO is a prerequisite for cyanide formation, and this pathway is exclusively catalyzed by CYP2E1. Finally, this data showed that inactivation of CYP2E1 is an efficient mechanism for the inhibition of cyanide formation in vivo. Inhibition of CYP2E1 may therefore constitute a beneficial therapeutic approach to counter human poisoning with AN. This approach may have the additional advantage of preventing AN metabolism to CEO and cyanide, which are implicated in the acute and chronic toxicities of this chemical.