Chem.-Biol. Intetuctions, 55 (1985) 317-326

Elsevier Scientific Publishers Ireland Ltd. 317

EFFECT OF DIETARY VITAMIN E ON METHYL ETHYL KETONE PEROXIDE DAMAGE TO MICROSOMAL CYTOCHROME P-450 PEROXIDASE*

MITSURU AND0 and AL L. TAPPEL**

Department of Food Science and Technology, University of California, Davis, CA 95616 (U.S.A.)

(Received May 24th. 1985) (Revision received July 1st. 1985) (Accepted July 2nd, 1985)

SUM MARY

The effect of dietary vitamin E on in vivo and in vitro damage by methyl ethyl ketone peroxide (MEKP) to cytochrome P-450 and its associated enzymatic activity was studied. In vivo, MEKP damaged microsomal cytochrome P-450 and cytochrome P-450-mediated peroxidases in vitamin Edeficient rat liver. Dietary vitamin E treatment of rats protected the microsomal enzymes from peroxide damage. In vitro, the extent of MEKP inhibition was different for tetramethylphenylenediamine (TMPD)-peroxi- dase. NADH-peroxidase, and aminopyrine demethylase. In vitro addition of MEKP induced production of more thiobarbituric acid reacting substances (TBABS) in liver microsomes from vitamin Edeficient rats than from vitamin Esupplemented rats. When NADH and/or NADPH were supplied as reductants of MEKP, the inhibition of aminopyrine demethylase activity and the generation of TBABS by added MEKP were markedly reduced. In vivo, adequate levels of vitamin E and of NADH and NADPH are probably necessary to provide important protection to the endoplasmic reticulum during metabolism of toxic organic peroxides, such as MEKP.

Key wonls: Vitamin E - Methyl ethyl ketone peroxide - Cytochrome P-450 peroxidase

*This research was supported by NIH grant AM-09933 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases.

**To whom correspondence should be sent. Abbreviations: MEKP, methyl ethyl ketone peroxide; TBARS, thiobarbituric acid reacting

substances; TMPD, tetramethylphenylenediamine.

0009-2797/85/$03.30 0 1985 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland

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INTRODUCTION

MEKP is used as a curing agent for polyester resins, and as a cross-linking agent in the production of other polymers [l]. MEKP can enter the body via inhalation and ingestion, or through skin or eye contact. When administered to experimental animals orally, intraperitoneally, or by inhalation, MEKP causes mild liver damage and hyperemia of lung, with resulting petechial or gross hemorrhage. MEKP also can severely irritate the eyes and skin, and can produce various types of malignant tumors [2-51.

MEKP is a substrate for microsomal cytochrome P-450 peroxidase, which includes NADH-peroxidase and NADPH-peroxidase activities, and it is an irreversible inhibitor of these enzymes [6]. There are two mechanisms by which MEKP can inhibit cytochrome P-450 peroxidase and microsomal monooxygenases: it can react directly with enzymes to covalently bind to their binding and reactive sites, or it can function as a potent inducer of lipid peroxidation. Mediation of lipid peroxide formation within the microsonies by MEKP can greatly inhibit the microsomal enzyme activities. This paper describes in vivo and in vitro studies of the effects of MEKP on cytochrome P-450 peroxidase in microsomes prepared from livers of rats fed vitamin Edeficient or vitamin Esupplemented diets, and treated or not treated with phenobarbital, an inducer of microsomal cytochrome P-450. MEKP-mediated lipid peroxidation of microsomes, and the inhibitory effect of MEKP on the cytochrome P-450 monooxygenase system were studied. The in vitro protection of microsomes by NADH and NADPH against MEKP-induced peroxidative damage was shown.

MATERIALS AND METHODS

Materials NADH, NADPH and phenobarbital were purchased from Sigma Chemical

Company, and MEKP and cumene hydroperoxide from Polysciences. All other chemicals were of the highest purity commercially available.

Animals, diets and treatments Male Sprague-Dawley rats were obtained from Simonsen Laboratories,

Inc. at 3 weeks of age, and were housed at 21-24°C with a 14-h light and 10-h dark cycle. Rats were fed for 6 weeks either a basal vitamin Edeficient diet [7] (Teklad Test Diets) or the same diet supplemented with 40 I.U. dl-a- tocopheryl acetate/kg. Each dietary group was divided into 4 treatment groups, as shown in Table I. Phenobarbital was dissolved in 1 NNaOH, adjusted to pH 9.5, and administered i.p. to rats at a dose of 80 mg/kg body wt. on 3 successive days prior to injection of MEKP. The phenobarbital solution was prepared fresh daily. Groups of vitamin Edeficient and vitamin Esupplemented rats were injected i.p. with 21.7 (one-third of the LDSO) and with 32.5 mg (one-half of the LD& of MEKP in tributyrimkg body wt.,

319

respectively. The rats were fasted for 24 h, anesthetized with ether, and killed by severing the main abdominal artery.

Prepamtion of microsomes Microsomes were prepared from rat liver according to the modified method

of Noshiro and Omura [8]. The livers were thoroughly perfused in situ, from the heart, with cold 0.9% NaCl. The perfused livers were homogenized with 9 volumes of 0.25 M sucrose, 10 n-&l Tris-HCl buffer (pH 7.5) and 1 mM EDTA in a Potter-Elvehjem glass-Teflon homogenizer. The homogenate was centrifuged at 9OOxg for 10min and then at 10000~ g for 15min. The resulting supernatant was centrifuged at 92 000 X g for 70 min. The microsomes were suspended in 0.15 M KC1 and 1 n-M EDTA, and centrifuged at 92000 x g for 70 min. The washed microsomes were suspended in 0.15 M KCl, 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA, and were stored immediately in liquid nitrogen. Cytochrome P-450 and cytochrome b, hemoprotein in the liver microsomes were measured the same day.

Andy tical methods Cytochrome P-450 and cytochrome b, were determined according to the

methods of Omura and Sato [9] and Estabrook and Werringloer [lo], using extinction coefficients of 91 n&I-’ cm-’ (between 490 and 450nm) and 185 mM’ cm-’ (between 426 and 409 nm), respectively. The concentration of microsomes for assay of cytochromes was 2mg protein/ml. Protein was measured by a Coomassie Blue dye-binding assay [ 111, using ovalbumin as a standard, TBARS were determined according to the modified method of Ohkawa et al. [12].

Enzyme assays Aminopyrine N-demethylase was assayed as described by Orrenius [13],

using 2 mg of microsomal protein/ml, 2mM NADH, 5mM MgC12, 5mM aminopyrine and 2 n-&I NADPH at 37°C for 5 min. All reagents contained 0.625 mM 5’-AMP to prevent breakdown of NADPH by microsomal phos- phatase [ 141.

TMPD oxidation activity was performed as described by O’Brien and Rahimtula [15], using 8OmM Tris-HCl (pH 7.5), 1 n-&l EDTA, 0.4mg of microsomal protein/ml, 1 mM TMPD, 0.1 mM sodium azide and 0.5 mM cumene hydroperoxide substrate. Reaction rates were corrected for TMPD oxidation in the absence of cumene hydroperoxide, and were measured by following the rate of Wurster’s blue free-radical formation at 610 nm, using an extinction coefficient of 11.6 n-&I-’ cm-‘.

NADH-peroxidase activity [16] was measured using 8OmM sodium phosphate buffer (pH 7.5), 1 mg microsomal protein/ml, 0.15 mM NADH, and 0.5 mM cumene hydroperoxide substrate. Reaction rates were corrected for NADH oxidation in the absence of cumene hydroperoxide, and were calculated using an extinction coefficient of 6.22 n&I-’ cm-‘.

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In vitro inhibition of cytochrom P-450 peroxidase by MEKP The in vitro inhibitory effects of MEKP on the enzymatic activities of

cytochrome P-450 were determined by measurement of TMPD-peroxidase, NADH-peroxidase, and aminopyrine demethylase. Microsomes were prepared from livers of vitamin Edeficient and vitamin Esupplemented rats. The pre-assay incubation mixtures consisted of 0.15 M KCl, 10 mM ‘lkis-HCl (pH 7.5), 1 mM EDTA, and 6 mg microsomal protein/ml. MEKP was added at concentrations of 25-300 PM. EDTA was included to prevent formation of free radicals by transition metal-catalyzed decomposition of MEKP. To measure the inhibition of TMPD-peroxidase and NADH-peroxidase by MEKP, the reaction mixtures were maintained at 24°C for O-20 min; then equal volumes of the mixtures were placed into sample and reference cuvettes, microsomal protein was appropriately adjusted and the cytochrome P-450 peroxidase activities were measured using the methods described above, with cumene hydroperoxide as substrate. Reaction rates were corrected for absorbance change in the absence of cumene hydroperoxide.

To measure the inhibition of aminopyrine demethylase activity by MEKP, the pre-assay incubation mixtures were maintained at 24°C for 0 to 20 min. To determine the effect of reductant on MEKP-induced damage, 2 mM NADH was added to the incubation mixtures. After incubation, microsomal protein was adjusted to 2mglm1, and aminopyrine demethylase activity was measured using the method described above. Reaction rates were corrected for formaldehyde formation in the absence of aminopyrine.

To measure the destruction of cytochrome P-450, MEKP was added to microsomal suspensions at concentrations of 300 PM. The incubation mixtures consisted of 0.15 M KCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 6 mg microsomal protein/ml. After being held at 24°C for 0 to 20 mm, reduced glutathione was added at a final concentration of 1.2 PM to reduce unreacted MEKP. Equal volumes of the mixtures were placed into sample and reference cuvettes, microsomal protein was adjusted to 2 mg protein/ml, and cytochrome P-450 was measured as described above.

To measure MEKP-mediated lipid per-oxidation, MEKP was added to microsomal suspensions at concentrations of 100-300 PM. The incubation mixtures consisted of 0.15 M KCl, 10 mM Tris-HCl (pH 7.5), 1 mM KDTA, and 6 mg microsomal protein/ml. To determine the effect of reductants on MEKP-generated lipid peroxidation, 2 mM NADH and 2 mM NADPH were added to the incubation mixtures. After being held at 24°C for O-20 min, TBARS were measured [ 121.

Statistical evaluation The significance of differences between any two groups of data was

determined by the Student’s t-test. P-values co.05 were considered significant.

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TABLE I

EFFECT OF VITAMIN E, PHENOBARBITAL AND MEKP ON CYTOCHROME P-450, CYTOCHROME P-450 PEROXIDASFS AND GLUTATHIONE S-TRANSFERASE IN RAT LIVER MICROSOMES

Methods are described in the text. Values are mean +S.D. The rats were fed a vitamin

Edeficient diet (OE) or a diet supplemented with 40 I.U. dl-a-tocopheryl acetate/kg (40E).

Diet Treatment

OE None

OE MEKP OE Phenobarbital OE Phenobarbital

+MEKP 40E None 40E MEKP

40E Phenobarbital 40E Phenobarbital

+MEKP

Cytochrome

P-450 (nmol/mg prot.)

0.28*0.02

0.23 f 0.01” 0.48*0.07b 0.39i0.07b

TMPD peroxidase

(nmol oxidized/ mg prot./min)

29*2.8 24k3.4" 65k9.1” 58zt 12b

NADH- peroxidase

(nmol oxidized/ mg prot./min)

21 f 1.3

17 i 2.7” 43i7.6'1 32xk5.2'1

Glutathione S-transferase (nmol product/

mg prot./min)

350*52 312~1~32 59 1 f 84” 7 13 f 109”

0.37 *to.04 30*3.7 21zt4.0 391*62 0.33zto.03 27 i3.3 18*3.0 436*40

0.45*0.04" 63i4.1d 39 f 1.9d 620*23d 0.41*0.07 71*16d 41i2.5" 552*42d

“Significantly lower than OE group with no treatment (P < 0.05). ‘Significantly higher than the OE group with no treatment (P < 0.05). ‘Significantly higher than the OE group with no treatment (P < 0.05). “Significantly higher than the 40E group with no treatment (P < 0.05).

RESULTS

In vivo effects of MEKP and phenobarbital on mt liver hemoprotein, cytochrome P-450 peroxidase, glutathione peroxidxse and glutathione S- tmnsfemse

In vivo damage to cytochrome P-450 peroxidase by MEKP was studied using microsomes prepared from vitamin Edeficient and vitamin E supplemented rats, some of which had been pretreated with phenobarbital to induce cytochrome P-450 peroxidase (Table I). Cytochrome P-450 and the activities of NADH-peroxidase and TMPD-peroxidase from vitamin E deficient rats not treated with phenobarbital were decreased significantly by MEKP treatment of rats. MEKP did not damage microsomal cytochrome P-450, cytochrome bti, NADH-peroxidase, or TMPD-peroxidase in either vitamin Esupplemented rats or in vitamin Edeficient rats treated with phenobarbital. Data for cytochrome bg are not shown as there were no significant differences among the groups. Phenobarbital treatment alone increased microsomal cytochrome P-450 and the associated activities of NADH-peroxidase and TMPD-peroxidase in both dietary groups of rats.

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TIME (MINI

Fig. 1. In vitro effect of 300 FM MEKP on cytochrome P-450 and NADH-peroxidase as a

function of incubation time. 0, cytochrome P-450; A, NADH peroxidase from vitamin Ekdeficient microsomes; 0, cytochrome P-450; A, NADH-peroxidase from vitamin E supplemented microsomes.

GSH-S-transferase (Table I) and GSH-peroxidase activities in the soluble fraction of liver from the various groups of rats were measured. MEKP treatment did not inhibit these enzymes in either vitamin Edeficient or vitamin Esupplemented rats; however, phenobarbital treatment induced GSH-S-transferase activity in both dietary groups of rats. The mean activity of glutathione peroxidase was 273 nmol NADPH oxidized/min/mg protein.

In vitro effect of MEKP on cytochrome P-450 and cytochrome P-450 peroxidase

The in vitro destructive effect of MEKP on cytochrome P-450 increased as a function of incubation time up to 20 min (Fig. 1). Microsomal cytochrome P-450 from vitamin Edeficient rats was damaged to a greater extent by MEKP than was that from vitamin Esupplemented rats. For vitamin E-supplemented microsomes, MEKP treatment in vitro resulted in a greater percentage loss of NADH-peroxidase activity (approx. 75% loss by 20 min) than of cytochrome P-450 protein (approx. 50% loss by 20 min). For vitamin Edeficient microsomes, the cytochromk P-450 protein and the associated NADPH-peroxidase activity were both decreased approx. 80% by MEKP treatment in vitro. MEKP inhibition of NADH-peroxidase from vitamin Edeficient and vitamin Esupplemented rats was similar.

MEKP inhibited liver microsomal TMPD-peroxidase from vitamin E deficient and vitamin Esupplemented rats to about the same extent (Fig. 2). In vitro inhibition increased as a function of time, and was dependent upon the concentration of MEKP. For vitamin Esupplemented microsomes, TMPD-peroxidase was damaged to a greater extent (approx. 80% by 20 min) by 300 PM MEKP than was cytochrome P-450 protein (approx. 50% by 20 min as shown in Fig. 1). For vitamin E-deficient microsomes, the protein and the enzyme activity were decreased by approx. 80% after 20 min incubation with 300 PM MEKP.

MEKP inhibition of cytochrome P-450-mediated aminopyrine demethylase activity was dependent upon the concentration of MEKP (Fig. 3). Aminopyrine demethylase in vitamin Edeficient microsomes was more

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Fig. 2. Effect of MEW on TMPD-perotidase activity in microsomes prepared from (A) vitamin E-supplemented and (B) vitamin E-deficient rats. 0, 25 PM MEKF’; n , 100 PM MEKC A,

300 FM MEKP.

Fig. 3. Effect of MEKP in the presence and absence of NADH on inhibition of aminopyrine

Ndemethylase in microsomes prepared from (A) vitamin Esupplemenkd and (B) vitamin Edeficient rata. 0, 25 PM MEKF’; 0, 25 PM MEKF’ and 2 mM NADH; A, 100 PM MEKP; A,

100 PM MEKP and 2 mM NADH; n , 300 PM MEKP.

sensitive to inhibition by MEKP than was the enzyme in vitamin E supplemented microsomes. When NADH was supplied exogenously as a reductant, the extent of inhibition was decreased.

In vitro initiation of lipid per-oxidation by MEKF’ The effect of dietary vitamin E on inhibition of lipid peroxidation induced

in vitro by MEKP was determined. MEKP induced formation of more TBARS in microsomes from vitamin Edeficient rats than from vitamin E supplemented rats (Fig. 4). The production of TBARS was dependent upon the concentration of MEKP. When NADH and NADPH were supplied as reductant, the generation of TBABS by MEKP was decreased.

DISCUSSION

Lipid peroxides can damage hepatic endoplasmic reticulum and inhibit some microsomal enzymes [ 17-191. Cytochrome P-450, the terminal oxidase of the hepatic microsomal monooxygenase system, is one of the enzymes most sensitive to lipid peroxide damage [20]. MEKP is a substrate for cytochrome P-450 peroxidase, an enzyme that may play an important role in metabolism of lipophilic organic peroxides. MEKP appears to immediately

TIME (MINI Fig. 4. Effect of MEKP on TBARS production in rnicrosomes prepared from (A) vitamin Esupplemented and (B) vitamin E-deficient rata. V, no addition; 0, 2mM NADH and 2mM

NADPH; 0. 100 FM MEKP; 0, 100 PM MEKP, 2mM NADH and 2mM NADPH; A, 200 PM MEKP; A, 200 PM MEKP, 2 mM NADH and 2 mM NADPH; l , 300 GM MEKP; U,

300 PM MEKP, 2 mM NADH and 2 mM NADPH.

bind to microsomal cytochrome P-450 peroxidase, and to cause irreversible inhibition of its peroxidase activities [6].

Since vitamin Edeficiency greatly potentiated the toxicity of MEKP to rats [21,22), in the present study vitamin Edeficient and vitamin Esupplemented rats were administered different doses of MEKP (one-third of the LDsO and one-half of the LD,,, respectively) in order to prevent deaths among the animals. Vitamin E was an effective in vivo protector against MEKP damage to microsomal cytochrome P-450, NADH-peroxidase, and TMPD-peroxidase. Phenobarbital treatment also protected the enzymes from damage by MEKP in vitamin Edeficient rats. Phenobarbital, an inducer of microsomal cytochrome P-450, induced liver cytochrome P-450 peroxidase and glutathione S-transferase. Induction of these enzymes may have significantly contributed to the in vivo protection that phenobarbital provided against MEKP damage to microsomes. Glutathione peroxidase, a soluble enzyme active against lipid hydroperoxides and hydrogen peroxide, was not induced by phenobarbital, and was unaffected by MEKP treatment of the rats. GIutathione peroxidase is relatively stable to treatment with MEKP, and MEKP is a substrate for the enzyme [23].

MEKP inhibited the various cytochrome P450-mediated enzyme activities to different extents. Although dietary vitamin E did not affect inhibition of TMPD-peroxidase by MEKP, it did provide slight protection to NADH- peroxidase, and it provided considerable protection to aminopyrine

demethylase. The sensitivities of cytochrome P-450 peroxidases to MEKP were previously shown [6] to be markedly different; NADPH-peroxidase was more resistant to damage by MEKP than was NADH-peroxidase. This investigation and the previous study [6] both suggest that MEKP may directly bind to cytochrome P-450 peroxidases to cause irreversible inhibition of their activities. MEKP generated more TBABS in microsomes from vitamin Edeficient rats than from vitamin Esupplemented rats, thus showing that one mechanism of action of this peroxide involves lipid per-oxidation reactions. It is probable that both MEKP and lipid peroxides generated by MEKP can damage cytochrome P-450 and cytochrome P-450- mediated aminopyrine demethylase activity When NADH was supplied as a reductant, the inhibition of aminopyrine demethylase by MEKP was greatly decreased. When NADH and NADPH were supplied as reductants, the generation of TBARS by MEKP was also greatly decreased. NADH is required for the activity of NADH-peroxidase [16], and NADPH is required for the activity of NADPH-peroxidase [24]; these enzymes were shown to metabolize MEKP [6]. Part of the protective effects of NADH and NADPH on microsomal protein may depend upon the activity of these enzymes. Vitamin E is presumed to protect microsomal components, including cytochrome P-450. Vitamin E sufficiency and adequate generation of the NADH and NADPH may be essential for the protection of microsomes from toxic lipophilic organic peroxides.

REFERENCES

1 C.S. Sheppard and O.L. Mageli. Peroxides and peroxy compounds, organic, in: K. Othmer

(Ed.), Encyclopedia of Chemical Technology, Vol. 17. Wiley-Interscience, New York. 1982, pp. 27-89.

2 National Institute for Occupational Safety and Health, Information profiles on potential

occupational hazards: MEKP, Report PB-276. 678, Rockville. MD. 1977. pp. 37-44. 3 M Sitting, Handbook of Toxic and Hazardous Chemicals, Noyes Publications, Park Ridge,

NJ, 1981. 4 SPI Bulletin, Commercial Organic Peroxide Toxicological Data, The Society of the Plastics

Industry, Inc., New York, 1982.

5 U.S. Environmental Protection Agency, Chemical Hazard Information Profile: MEKP, Washington, DC, 1979.

6 M. Ando and A.L. Tappel, Peroxide damage to enzymes: Methyl ethyl ketone peroxide damage to cytochrome P-450 peroxidase, Fed. Proc., 44 (1985) 1446.

7 H.H. Draper, J.G. Bergan, M. Chiu, A.S. Csallany and A.V. Boaro, A further study of the

specificity of the vitamin E requirement for reproduction, J. Nutr., 84 (1964) 395-400. 8 M. Noshiro and T. Omura, Immunochemical study on the electron pathway from NADH to

cytochrome P-450 of liver microsomes, J. Biochem.. 83 (1978) 61-77.

9 T. Omura and R. Sate, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem.. 239 (1964) 2370-2378.

10 R.W. Estabrook and J. Werringloer, The measurement of difference spectra: application to

cytochromes of microsomes, in: S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. LII, Academic Press, New York, 1978, pp. 212-220.

11 M Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254.

326

12 H. Ohkawa, N. Ohishi and K. Yagi, Assay for lipid. peroxides in animal tissues by

thiobarbituric acid reaction, Anal. Biochem., 95 (1979) 351-358. 13 S. Orrenius, On the mechanism of drug hydroxylation in rat liver microsomes, J. Cell Biol..

26 (1965) 7 13-723. 14 J. Werringloer. Assay of formaldehyde generated during microsomal oxidation reactions, in:

S. Fieischer and L. Packer (Eds.), Methods in Enzymology, Vol. LII, Academic Press, New York, 1978, pp. 297302.

15 P.J. O’Brien and A.D. Rahimtula, A peroxidase assay for cytochrome P-450, in: S. Fleischer

and L. Packer (Eds.), Methods in Enzymology, Vol. LII, Academic Press, New York, 1978, pp. 407-412.

16 E.G. Hrycay and P.J. O’Brien, Microsomal electron transport. II. Reduced nicotinamide adenine dinucleotide-cytochrome b, reductase and cytochrome P-450 as electron carriers in microsomal NADH-peroxidase activity. Arch. Biochem. Biophys., 160 (1974) 230-245.

17 A.M. Hruszkewycz, E.A. Glende, Jr. and R.O. Recknagel. Destruction of microsomal cytochrome P-450 and glucose-6-phosphatase by lipid extracted from peroxidized microsomes. Toxicol. Appl. Pharmacol., 46 (1978) 695-702.

18 E. Cadenas and H. Sies, Low level chemiluminescence of liver microsomal fractions initiated by tert-butyl hydroperoxide. J. Biochem., 124 (1978) 349-356.

19 P.J. O’Brien. Hydroperoxides and superoxides in microsomal oxidations, in: J.B.

Schenkman and D. Kupfer (Eds.), International Encyclopedia of Pharmacology and Therapeutics, Section 108: Hepatic Cytochrome P-450 Monooxygenase System, Pergamon Press, New York, 1982. pp. 567-586.

20 L.H. Jeffery, D. Nerland, D.R. el-Azbary and G.J. Mannering. Destruction of cytochrome P-450 by linoleic acid hydroperoxide, in: V. Ullrich. I. Roots, A. Hildebrandt. R.W. Estabrook and A.H. Conney (Rds.), Microsomes and Drug Oxidations, Pergamon Press, New

York, 1977, pp. 323-330. 21 L.A. Herschberger and A.L. Tappel. Effect of vitamin E on pentane exhaled by rats treated

with methyl ethyl ketone peroxide, Lipids, 17 (1982) 686-691.

22 R.E. Litov, L.C. Matthews and A.L. Tappel, Vitamin E protection against in vivo lipid peroxidation initiated in rats by methyl ethyl ketone peroxide as monitored by pentane.

Toxicol. Appl. Pharmacol., 59 (1981) 96106. 23 R.A. Condell and A.L. Tappel, Evidence for suitability of glutathione peroxidase as a

protective enzyme: studies of oxidative damage, renaturation and proteolysis. Arch.

Biochem. Biophys., 223 (1983) 407-416.

24 E.G. Hrycay and P.J. O’Brien. Microsomal electron transport. I. Reduced nicotinamide

adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity, Arch. Biochem. Biophys., 157 (1973) 7-22.