In Vivo Production of Nitric Oxide after Administration of

Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Science, National Institutes of Health, P.O. Box 12233, Research ...
0 downloads 0 Views 141KB Size
Download PDF
952

Chem. Res. Toxicol. 1999, 12, 952-957

In Vivo Production of Nitric Oxide after Administration of Cyclohexanone Oxime Richard E. Glover,* Jean T. Corbett, Leo T. Burka, and Ronald P. Mason Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Science, National Institutes of Health, P.O. Box 12233, Research Triangle Park, North Carolina 27709 Received April 1, 1999

Cyclohexanone oxime (CHOX), an intermediate used in the synthesis of polycaprolactam (Nylon-6), has been reported to be hematotoxic in Fischer rats. The in vivo metabolism of CHOX was found to release nitric oxide, which was detected in venous blood by electron paramagnetic resonance spectroscopy as the nitrosylhemoglobin complex. In vitro incubation of CHOX with venous blood resulted in the formation of the characteristic nitrosylhemoglobin complex, suggesting that the blood was a possible site for metabolism. Excessive nitric oxide production may, in part, contribute to the observed toxicity of CHOX.

Introduction Cyclohexanone oxime (CHOX)1 is primarily used as a captive intermediate in the synthesis of caprolactam, which is polymerized in the production of polycaprolactam (Nylon-6) fibers and plastics. Polycaprolactam (Nylon6) is used widely in clothing, carpeting, home furnishings, food packaging film, and molded plastics for automobiles and appliances. As a result, because of the large volume of production of CHOX, the potential for exposure to this chemical is high. Studies with CHOX have shown that the erythrocyte is the main target in vivo, causing hemolytic anemia with compensatory extramedullary erythropoiesis as well as hemosiderosis in the spleen (1, 2). CHOX and other oximes, including the related compound hydroxylamine, have been shown to elevate the level of methemoglobin and thiobarbituric acid-reactive substances and to deplete glutathione levels, indicative of oxidative stress, in human hemolysate, erythrocytes, or blood (3, 4). It is not well-understood whether the hematotoxic effects associated with CHOX are due to the parent compound per se or to its metabolites. In rats, CHOX is metabolized to its corresponding ketone and is further metabolized and excreted in urine as glucuronide conjugates (5). Whether hydroxylamine, a compound with strong oxidative capacity with regard to hemoglobin, is also formed and is ultimately responsible for the toxicity of CHOX remains to be proven. Systemically absorbed CHOX induces oxidative damage to the erythrocyte population of animals, resulting in hemolytic anemia which is accompanied by erythropoiesis (1). Subchronic exposure of rats to CHOX resulted in an increased level of methemoglobin formation along with dose-dependent decreases in the number of eryth* To whom correspondence should be addressed: Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709. Telephone: (919) 541-0495. Fax: (919) 541-1043. E-mail: [email protected]. 1 Abbreviations: CHOX, cyclohexanone oxime; CHO, cyclohexanone; HbNO, nitrosylhemoglobin complex; EPR, electron paramagnetic resonance; NO, nitric oxide; LPS, lipopolysaccharide; metHb, methemoglobin.

10.1021/tx990058v

rocytes, a decrease in hemoglobin content and lower hematocrit, and a concomitant increase in the level of circulating reticulocytes and nucleated erythrocytes (1, 2). Hematological disorders have been reported in humans exposed to CHOX, and dermatitis and skin sensitization may also be potential effects of occupational exposure (2). CHOX is rapidly absorbed after a single oral administration and completely excreted from the body, mainly in the urine, within 24 h (5). However, the site and mechanism of CHOX metabolism in vivo have yet to be fully elucidated. The aim of this work was to extend the scope of knowledge concerning the toxicological properties of CHOX, specifically, to determine the site and mechanism of metabolism in vivo. In this report, electron paramagnetic resonance (EPR) spectroscopy, also known as electron spin resonance (ESR), was used to show that CHOX is metabolized to nitric oxide (NO); specifically, we take advantage of the binding of NO to deoxyhemoglobin to give EPR spectra characteristic of the nitrosylhemoglobin complex (HbNO) at 77 K.

Materials and Methods Materials. CHOX was purchased from Aldrich Chemical Co. (Milwaukee, WI) and recrystallized in hexane prior to use. Deionized water was used as the vehicle for the preparations of the dosing solutions and as a control. Dosing solutions were freshly prepared just prior to use. Oximes structurally similar to CHOX have been demonstrated to be animal carcinogens. Due care should be taken. [1-14C]CHOX (specific activity of 6.85 mCi/ mmol) was obtained from NEN Research Products (Boston, MA). Radiochemical analysis by HPLC indicated that the ratio of [14C]cyclohexanone to [14C]CHOX was 0.035. [15N]CHOX was prepared using the method of Adams et al. (6) and recrystallized in hexane prior to use (mp 93.5-94 °C). Animal Preparations. Male Fischer rats (F344) used throughout the experiments were obtained from Charles River Laboratories (Raleigh, NC). All animals were acclimated for at least 7 days prior to the start of the experiments. Rats were fasted overnight prior to the oral administration of a single dose of 200 mg/kg CHOX for the time course studies. The rats were anesthetized with sodium pentobarbital (Nembutal, 50 mg/kg, ip); then 400 µL of blood was obtained via the vena cava at the

This article not subject to U.S. Copyright. Published 1999 by the American Chemical Society Published on Web 09/04/1999

Metabolism of Cyclohexanone Oxime indicated times. Each time point represents data from three animals. The blood was then transferred into EPR quartz tubes (3 mm i.d. × 4 mm o.d.), frozen immediately in liquid nitrogen, and stored at -70 °C prior to EPR analysis. For the doseresponse study, rats were treated with a CHOX dose that ranged from 50 to 200 mg/kg. Two hours after administration, the rats were sacrificed and venous blood was collected from the abdominal vena cava. The liver was excised and immediately frozen on solid carbon dioxide and stored at -70 °C prior to EPR analysis. In Vitro Incubations. Fresh blood was collected from the abdominal vena cava of an untreated rat in a vaccutainer containing EDTA. For the time course of in vitro metabolism of CHOX to NO, venous blood was incubated with 2 mg/mL CHOX for a time period ranging from 30 min to 3 h at 37 °C in sealed glass tubes. After incubation, 1 mL of blood was frozen immediately in liquid N2 for EPR measurements. Similar experiments in which saline was used instead of cyclohexanone oxime were carried out as controls. To verify that the NO that was produced was due to metabolism of CHOX and not leukocyte stimulation, LPS (200 µg/mL) was added to whole blood and incubated as for the CHOX-treated blood. The in vitro dose dependency of the HbNO signal on CHOX concentration was determined as follows. Venous blood was incubated with CHOX (0-2 mg/kg) for 2 h at 37 °C. After incubation, blood was transferred into EPR quartz tubes, frozen immediately in liquid nitrogen, and stored at -70 °C. A comparison of the HbNOforming capacity of CHOX with that of hydroxylamine was also carried out in a similar manner. Stabilty of [14C]CHOX. To determine the stability of CHOX in blood and plasma, fresh blood from the abdominal vena cava from three untreated rats was collected as described above, pooled, and divided into portions. One portion was centrifuged to give plasma. Phosphate-buffered saline (1 mL) containing 14Clabeled CHOX was added to 9 mL of plasma or whole blood to give a final concentration of 1.5 mg/ml and 2 µCi/mL. The blood and plasma were incubated at 37 °C in a water bath. Aliquots were removed for HPLC analysis and for histopathological examination after 5 min and 1, 3, and 4 h. EPR Analysis. All EPR measurements were carried out on a Bruker ESP300 spectrometer operated at 9.5 GHz with a 100 kHz modulation frequency at liquid nitrogen temperatures with samples held in a quartz finger dewar. Typical spectrometer settings were as follows: 20 mW power, 4 G modulation amplitude, 1.31 s time constant, 1342 s scan time, and 400 or 3500 G scan range. The EPR signal from Cr3+ in MgO was used as a g-value marker (g ) 1.9800 ( 0.0006) (7). HPLC Analysis. The HPLC system consisted of two Waters (Milford, MA) pumps, an automated system controller, and a Waters model 481 UV detector set at 260 nm. Radiochemical detection was accomplished using an IN\US (Tampa, FL) betaRam flow detector. A 4.6 mm × 250 mm Rainin (Varian Associates, Walnut Creek, CA) C18 5 µm column was used with a linear solvent gradient of 100% 50 mM KH2PO4 (pH 7.0) to 50% phosphate buffer/50% acetonitrile over the course of 28 min.

Results The EPR spectra of venous blood from rats treated with 200 mg/kg CHOX (intragastrically) exhibited signals characteristic of the pentacoordinated deoxyhemoglobinnitrosyl complex (8), indicative of NO generation (Figure 1). The EPR spectra exhibit a well resolved triplet structure at g ) 2.011 with a hyperfine coupling constant of 17.4 G due to the 14N assigned to the nitrosylhemoglobin (HbNO) complex (Figure 1A). The broad signal at g ) 2.06 has been attributed to Cu2+ arising from the serum protein ceruloplasmin (9). Figure 1B shows the EPR spectrum obtained under the same conditions, except [15N]CHOX was administered. A doublet hyperfine structure with a hyperfine coupling con-

Chem. Res. Toxicol., Vol. 12, No. 10, 1999 953

Figure 1. Formation of the nitrosylhemoglobin complex in vivo. Two hours after the 200 mg/kg oral dose of CHOX had been administered, the rats were anesthetized and venous blood was collected via the abdominal vena cava. The blood was then transferred into EPR quartz tubes, frozen immediately in liquid nitrogen, and stored at -70 °C prior to EPR analysis. (A) The blood from [14N]CHOX-treated rats exhibits a resolved triplet structure at g ) 2.011 with a hyperfine coupling constant (14NAz) of 17.4 G due to the 14N assigned to the nitrosylhemoglobin (HbNO) complex. (B) The blood from [15N]CHOX-treated rats exhibits a doublet hyperfine structure and a hyperfine coupling constant (15NAz) of 25.0 G which is attributed to [15N]HbNO. (C) The blood from untreated rats. The broad signal at g ) 2.06 has been attributed to Cu2+ arising from the serum protein ceruloplasmin.

stant of 25.0 G was attributed to [15N]HbNO formation and is the result of the change in nuclear spin from 1 (14NO) to 1/2 (15NO). The 15N isotope experiments clearly demonstrate that the origin of the NO was the NOH moiety of CHOX. In the time course experiments, the formation of HbNO could be detected as early as 30 min after oral administration and reached the maximal level after 2 h (Figure 2). The level of production of HbNO from CHOX metabolism increased in a dose-dependent manner from 50 to 200 mg/kg (Figure 3). There were no detectable nitrosyl complexes in the livers of rats treated with up to 200 mg/kg CHOX and sacrificed 2 h later (data not shown). The status of the liver cytochromes P450 and P420 was investigated by recording EPR spectra over a scan range of 1000 G. The known EPR signals of low-spin ferric ion of both cytochrome P450 and P420 exhibited similar resonance peaks at g ≈ 2.43, g ≈ 2.25, and g ≈ 1.92 (10); we observed these cytochrome species at g ≈ 2.43, g ≈ 2.26, and g ≈ 1.92 in both normal and CHOX-treated livers (Figure 4). We found that the ferric low-spin heme groups (g ≈ 2.26) indicative of cytochrome P450 or P420 did not vary with the dose of CHOX that was administered (Figure 5). Interestingly, the intensity of the g ≈ 2.0 unidentified signal increased in a dose-dependent manner over the range that was studied (Figure 5). In vitro incubation of whole blood with 2 mg/mL CHOX resulted in the formation of the characteristic deoxyHbNO spectrum, indicative of NO generation (Figure 6A). Figure 6B shows the EPR spectrum obtained under the

954

Chem. Res. Toxicol., Vol. 12, No. 10, 1999

Figure 2. Time course of the relative concentration of the nitrosylhemoglobin complex in venous blood after a single 200 mg/kg oral dose of CHOX. Blood was obtained via the vena cava at the indicated times. Blood was transferred into EPR quartz tubes, frozen immediately in liquid nitrogen, and then stored at -70 °C prior to EPR analysis. EPR measurements were carried out as described in Materials and Methods. The EPR spectra were double-integrated, and the background signal was subtracted to give relative concentrations of HbNO. Each individual point represents the mean ( standard error of the mean from three animals.

Figure 3. Dose-dependent increase in the intensity of the HbNO signal observed in venous blood 2 h after oral administration of 0-200 mg/kg CHOX. Venous blood was collected, transferred into an EPR quartz tube, frozen immediately in liquid nitrogen, and then stored at -70 °C prior to EPR analysis. EPR measurements were carried out as described in Materials and Methods. The EPR spectra were double-integrated, and the background was subtracted to give relative concentrations of HbNO. Each point represents the mean ( standard error of the mean from four individual animals.

same conditions, except [15N]CHOX was used with the expected change in the spectrum. The time course of the formation of HbNO in vitro follows a profile very similar to that observed in vivo (data not shown). The level of formation of HbNO from CHOX in vitro increased in a dose-dependent manner over the range of concentrations that were used (data not shown). To further verify that

Glover et al.

Figure 4. Ferric cytochrome P450 or P420 resonance in rat livers at 77 K. (A) Representative EPR spectrum obtained from the liver of a rat treated with 200 mg/kg CHOX. (B) EPR spectrum obtained from control rat liver. Resonance features at g ≈ 2.43, g ≈ 2.26, and g ≈ 1.92 are signals attributed to low-spin ferric cytochromes P450 and P420. EPR measurements were carried out as described in Materials and Methods.

Figure 5. Summary of the peak intensities of liver g ) 2.26 low-spin ferric cytochromes P450 and P420 and the corresponding dose-dependent increase in the intensity of the g ) 2 unidentified resonance. The liver was excised from anesthetized rats treated with 0-200 mg/kg CHOX after 2 h. All samples were immediately frozen in liquid nitrogen and stored at -70 °C prior to EPR analysis. EPR measurements were carried out as described in Materials and Methods over a 1000 G scan range. Each point represents the mean ( standard error of the mean from two sets of duplicate experiments.

the HbNO signal observed in vitro was from CHOX metabolism and not from activated leukocytes, whole blood was incubated with 200 µg/mL LPS. Incubation with LPS did not result in any observable HbNO signal (data not shown), further supporting our observations that CHOX metabolism in the blood leads to HbNO formation. The formation of methemoglobin (metHb) was determined in vitro by measuring the relative intensity of the

Metabolism of Cyclohexanone Oxime

Figure 6. In vitro formation of the nitrosylhemoglobin complex. Blood was obtained from anesthetized rats via the abdominal vena cava and incubated with 2 mg/mL CHOX for 2 h at 37 °C. Blood was then transferred into EPR quartz tubes, frozen immediately in liquid nitrogen, and stored at -70 °C prior to EPR analysis. EPR measurements were carried out as described in Materials and Methods. (A) The blood incubated with [14N]CHOX exhibits the resolved triplet structure at g ) 2.011 with a hyperfine coupling constant (14NAz) of 17.4 G due to the 14N assigned to the nitrosylhemoglobin (HbNO) complex. (B) The blood incubated with [15N]CHOX exhibits a doublet hyperfine structure and a hyperfine coupling constant (15NAz) of 25.0 G which is attributed to [15N]HbNO. (C) EPR spectrum of blood incubated with saline. The broad signal at g ) 2.06 has been attributed to Cu2+ arising from the serum protein ceruloplasmin.

Chem. Res. Toxicol., Vol. 12, No. 10, 1999 955

Figure 7. Summary of the dose-dependent increase in the peak intensity of the g ) 6 high-spin methemoglobin (Fe3+). Blood was obtained from anesthetized rats via the abdominal vena cava and incubated with 0-2 mg/mL CHOX for 2 h at 37 °C (b). Rats were treated with 0-200 mg/kg CHOX, and venous blood was obtained after 2 h (9). All samples were immediately frozen in liquid nitrogen and stored at -70 °C prior to EPR analysis. Samples were run within 7 days of collection to minimize the extent of hemoglobin autoxidation. EPR measurements were carried out as described in Materials and Methods, except over a 1000 G scan range. Each point represents the mean ( standard error of the mean from four experiments.

low-field g ≈ 6 high-spin MetHb peak after incubation for 2 h in venous blood with CHOX. The relative intensity of the g ≈ 6 signal increased in a dose-dependent manner over the range of 0-2 mg/mL CHOX (Figure 7). Our observations are consistent with the oxidative properties of CHOX on red blood cells in vitro previously reported (3). However, in vivo, there was no detectable difference in metHb concentrations in blood between control and CHOX-treated animals (Figure 7). The lack of a dosedependent increase in the metHb concentrations in vivo may be attributed to the action of methemoglobin reductase, which may be compromised in vitro. A possible mechanism for HbNO formation may be hydrolysis of the oxime moiety to form the labile NO source, hydroxylamine. We therefore sought to compare in vitro the HbNO-forming capacity of CHOX with that of hydroxylamine on a molar basis. We found that hydroxylamine was approximately 40-fold more potent than CHOX in forming HbNO as determined by EPR in vitro (data not shown). However, in vivo, when hydroxylamine was administered via oral gavage, it was found to be approximately 10-fold more potent than CHOX (data not shown). Therefore, only relatively low concentrations of hydroxylamine need be formed from CHOX in the blood to account for its HbNO-forming capacity. Additional evidence for CHOX metabolism in blood was obtained by incubating [14C]CHOX with blood or plasma. CHOX was essentially unchanged in plasma over 4 h at 37 °C. However, in whole blood there is evidence for conversion of CHOX to cyclohexanone (CHO) (Figure 8).

Figure 8. Stability of CHOX in whole blood (0) and plasma (9). Fresh blood from the abdominal vena cava from three untreated rats was collected as described in Materials and Methods, pooled, and divided into portions. One portion was centrifuged to give plasma. Phosphate-buffered saline (1 mL) containing 14C-labeled CHOX was added to 9 mL of plasma or whole blood to give final concentrations of 1.5 mg/mL and 2 µCi/ mL. The blood and plasma were incubated at 37 °C in a water bath. Aliquots were removed for HPLC analysis after 5 min and 1, 3, and 4 h.

Discussion In this study, we have provided evidence for CHOX metabolism to NO in Fischer rats. The data presented

956

Chem. Res. Toxicol., Vol. 12, No. 10, 1999

here show EPR spectra representative of a five-coordinate nitrosyl-heme complex derived from NO binding to deoxyhemoglobin. In particular, the three-line hyperfine coupling at g ) 2.011, which results from the HbNO complex in which NO is bound to the heme iron of the R subunit, is characteristic of HbNO in venous blood (8). The measured hyperfine coupling constant (17.4 G) and g value (2.011) are in good agreement with values reported in the literature and are considered to be a fingerprint for in vivo NO production (11). Furthermore, the 15N isotope experiments confirmed that the NO that was produced did originate from the NOH moiety of the CHOX. However, the exact mechanism(s) of CHOX metabolism for producing NO in vivo remains to be elucidated. Jousserandot and co-workers (12) have shown that rat liver microsomes catalyzed the oxidative cleavage of Cd N(OH) to yield NO and the corresponding aldehyde or ketone in a reaction that was dependent on cytochrome P450 and required NADPH and O2. If CHOX were metabolized in the liver to release NO, it might then be possible to detect it as the heme (Fe2+)-nitrosyl complex of P450 or P420. However, when the liver was studied, there were no EPR-detectable nitrosyl-heme complexes. The detected cytochrome P450 or P420 did not vary with dose as determined by measuring the ferric low-spin signal at g ≈ 2.26. If CHOX were metabolized to NO in the liver, then it would be expected that the NO that is produced would reduce some of the ferric cytochrome P450 or P420 to the ferrous form with a concomitant decrease in the intensity of the ferric g ≈ 2.26 EPR signal and formation of the ferrous nitrosyl-cytochrome P450 or P420 complex (11). Interestingly, we found that the concentration of the g ≈ 2 unidentified radical, generally attributed to a flavanoid semiquinone radical in the liver (13), increased in a dose-dependent manner over the range of CHOX concentrations used throughout the study. We are unsure at present about how to interpret the relevance of the increased level of g ≈ 2 unidentified radical in the liver. We have observed that 14C-labeled CHOX is slowly converted to [14C]CHO in whole rat blood, but not in plasma. Over the 4 h incubation period, 6-7% of the CHOX was converted to CHO. A well-known phenomenon associated with treatment of erythrocyte with hydroxylamine in vitro (14) or exposure to oximes in vivo (1) is the formation of Heinz bodies. These consist of precipitated, oxidatively denatured hemoglobin (15). No Heinz bodies were observed in the erythrocytes from these incubations, indicating that the oxime is probably not directly responsible for their formation.2 In our experiments, the concentration of hydroxylamine produced from the hydrolysis of CHOX may not have been high enough to make sufficient oxidized hemoglobin to form observable Heinz bodies. Our data suggest that metabolism of CHOX to release NO may not occur in the liver in vivo. Palmen and Evelo (3) found that the oximes they studied, including CHOX and hydroxylamine, had no effect on isolated rat hepatocytes. However, it may be possible that CHOX is metabolized to CHO and hydroxylamine predominantly in liver and that hydroxylamine is then converted to NO in red blood cells to yield HbNO. We found that hydroxyl2 G. Travlos, National Institute of Environmental Health Sciences, unpublished observations.

Glover et al. Scheme 1. Proposed Metabolism of CHOX for Yielding HbNO in Red Blood Cells (RBCs)

amine in blood in vitro, in an equimolar ratio, was 40fold more potent than CHOX in forming HbNO. Others have found that oxidative stress induced by the oximes, as determined by TBARS formation and glutathione depletion, occurred only if erythrocytes were present (3). It was suggested that a factor present in the erythrocyte was necessary to induce oxidative stress and that metabolism of CHOX was not a limiting factor in erythrocyte toxicity (3). In vitro studies showed that incubation of venous blood with CHOX could also produce detectable nitrosylhemoglobin complexes, indicative of NO production, in a dose-dependent manner. Our data suggest that oxidative cleavage of the oxime moiety to NO may occur in the blood independent of liver metabolism. The partial hydrolysis of CHOX to hydroxylamine in blood may account for the HbNO and metHb formed in red blood cells in vitro (16). The extent of oxidation of hemoglobin to methemoglobin by CHOX increased in a dose-dependent manner as determined by the assessment of high-spin ferric heme at g ≈ 6. The oxidation of hemoglobin to methemoglobin by hydroxylamine in vitro is consistent with previous observations in the literature (3). However, the extent of methemoglobin formation in vivo did not increase after a single oral dose up to 200 mg/kg; this result may be attributed to the more effective action of methemoglobin reductase in vivo. We propose that hydroxylamine, a compound with strong oxidative capacity with regard to hemoglobin, may be formed as an intermediate and further metabolized to NO (Scheme 1). We have found that hydroxylamine is 40-fold more potent than CHOX in forming HbNO in vitro (data not shown). However, in vivo, when hydroxylamine was administered via oral gavage, it was found to be only approximately 10-fold more potent than CHOX (data not shown). Therefore, the partial hydrolysis of CHOX to hydroxylamine in the blood may account for the observed HbNO. The CHOX oxidative capacity reported in the literature may be attributed to hydroxylamine formation and, in part, to the NO that is produced.

References (1) Derelanko, M. J., Gad, S. C., Powers, W. J., Mulder, S., Gavigan, F., and Babich, P. C. (1985) Toxicity of cyclohexanone oxime. I. Hematotoxicity following subacute exposure in rats. Fundam. Appl. Toxicol. 5, 117-127. (2) Gad, S. C., Derelanko, M. J., Powers, W. J., Mulder, S., Gavigan, F., and Babich, P. C. (1985) Toxicity of cyclohexanone oxime. II. Acute dermal and subchronic oral studies. Fundam. Appl. Toxicol. 5, 128-136. (3) Palmen, N. G. M., and Evelo, C. T. A. (1998) Oxidative effects in human erythrocytes caused by some oximes and hydroxylamine. Arch. Toxicol. 72, 270-276. (4) Evelo, C. T. A., Spooren, A. A. M. G., Bisschops, R. A. G., Baars, L. G. M., and Neis, J. M. (1998) Two mechanisms for toxic effects of hydroxylamines in human erythrocytes: Involvement of free radicals and risk of potentiation. Blood Cells 24, 280-295. (5) Parmar, D., and Burka, L. T. (1991) Metabolism and disposition of cyclohexanone oxime in male F-344 rats. Drug Metab. Dispos. 19, 1101-1107.

Metabolism of Cyclohexanone Oxime (6) Adams, R., Johnson, J. R., and Wilcox, C. F., Jr. (1996) Carbonyl reactions of aldehyde and ketones. In Laboratory Experiments in Organic Chemistry, 5th ed., pp 217-223, The MacMillan Co., New York. (7) Low, W. (1957) Paramagnetic resonance and optical absorption spectra of Cr3+ in MgO. Phys. Rev. 105, 801-805. (8) Kosaka, H., Sawai, Y., Sakaguchi, H., Kumura, E., Harada, N., Watanabe, M., and Shiga, T. (1994) ESR spectral transition by arteriovenous cycle in nitric oxide hemoglobin of cytokine-treated rats. Am. J. Physiol. 266, C1400-C1405. (9) Hall, D. M., Buettner, G. R., Matthes, R. D., and Gisolfi, C. V. (1994) Hyperthermia stimulates nitric oxide formation: electron paramagnetic resonance detection of NO-heme in blood. J. Appl. Physiol. 77, 548-553. (10) Stern, J. O., Peisach, J., Blumberg, W. E., Lu, A. Y. H., and Levin, W. (1973) A low-temperature EPR study of partially purified, soluble ferric cytochromes P-450 and P-448 from rat liver microsomes. Arch. Biochem. Biophys. 156, 404-413. (11) Chamulitrat, W., Jordan, S. J., and Mason, R. P. (1994) Nitric oxide production during endotoxic shock in carbon tetrachloridetreated rats. Mol. Pharmacol. 46, 391-397.

Chem. Res. Toxicol., Vol. 12, No. 10, 1999 957 (12) Jousserandot, A., Boucher, J.-L., Henry, Y., Niklaus, B., Clement, B., and Mansuy, D. (1998) Microsomal cytochrome P450 dependent oxidation of N-hydroxyguanidines, amidoximes, and ketoximes: mechanism of the oxidative cleavage of their CdN(OH) bond with formation of nitrogen oxides. Biochemistry 37, 1717917191. (13) Van der Kraaij, A. M. M., Koster, J. F., and Hagen, W. R. (1989) Reappraisal of the e.p.r. signals in (post)-ischaemic cardiac tissue. Biochem. J. 264, 687-694. (14) Maile, J. B. (1982) Appendix-Methods. In Laboratory Medicine Hematology, 6th ed., pp 859-938, The C. V. Mosby Co., St. Louis, MO. (15) Jain, N. C. (1986) Hemolytic anemias of noninfectious origin. In Schalm’s Veterinary Hematology, 4th ed., pp 627-654, Lea and Febiger, Philadelphia, PA. (16) Stolze, K., and Nohl, H. (1989) Detection of free radicals as intermediates in the methemoglobin formation from oxyhemoglobin induced by hydroxylamine. Biochem. Pharmacol. 38, 3055-3059.

TX990058V