Life Sciences Vol . 19, pp " 1151-1160, Printed in the U .S .A .

Pergamon Press

1976 .

EFFECT OF DIETARY ANTIOXIDANTS AND PHENOBARBITAL PRETREATMENT ON~ MICROSOMAL LIPID PERORIDATION AND ACTIVATION BY CARBON TETRACHLORIDE Steve L . Taylorl and A1 L. Tappel Department of Food Science and Technology University of California, Davie,

California 95616

(Received in final form August 23, 1976) Summary The effect of the dietary antioxidants, vitamin E and selenium, and the effect of phenobarbital pretreatment on in vitro NADPH-dependent microsomal lipid peroxidation and the activation of micro somal lipid peroxidation by CC14 were studied. The rate of microsomal lipid peroxidation decreased as a function of dietary antioxidant, while the degree of CC14 activation increased . Phenobarbital pretreatment diminished the antioxidant inhibition of microsomal lipid peroxidation found with microsomes from rats fed high levels of antioxidant . Phenobarbital pretreatment lowered the aztent of lipid peroxidation as measured by malonaldehyde production but had little effect on the rate of lipid peroxidation as measured by oxygen uptake . The kinetics of lipid peroxidation and the atoichiometry of the reaction were assessed as a function of dietary antioxidant. Tha findings sugg®st that at low microsomal antioxidant concentrations, the lipid peroxidation reaction occurs at a maximal rate ~e~peadent upon some rata-limiting step, such as the reduction of Fe ,which is unaffected by CC1 4 addition . Conversely, at high microsomal antioxidant concentrations, the antioxidant termination reactions appear to determine the overall reaction rate . Modification of carbon tetrachloride (CC1 )-induced liver injury by dietary antioxidants, primarily vitamin E and seléaium, has been the subject of numerous investigations (1-13) . Hove and co-workers (1,2) first showed that vitamin E would protect animals from the lethal consequences of CC14 . Gallagher (3,4) demonstrated that sodium selenite would provide similar protection . Recknagel (14), Recknagel and Glende (15), and Slater (16) have used these studies along with many others to suggest that CC14 potentiates tonic lipid peroxidation processes . However, certain anomalous observations have created some controversy concerning the proposed free radical mechanism of CC1!, toxicity (8-11) . Some of the confusion stems from the variety of dosage patterns, routes of administration, and parameters of toxicity chosen by the various investigators . Obviously, feeding antioxidants over a relatively long period of time is the best method to attain a steady state level of these antioxidants in vivo . This report details the results of studies on the in vitro 1Preaent address : Food Hygiene Division, Department of Nutrition, Letterman Army Institute for Research, Presidio Saa Francisco, California 94129 . 1151

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CC14 activation of nicotinamide adenine diaucleotide phosphate, reduced form (NADPH)-dependent lipid peroxidation is microsomea isolated from animals fed The results provide partial various dietary levels of vitamin E and selenium . explanation for some of the anomalies observed is earlier studies . Materials and Methods Four groups of sixteen male Sprague-Dawley rats that weighed approximately 50 g each were fed for four weeks one of four special diets? The four diets varied only in the amounts of the dietary antioxidants, DL-a-tocopherol acetate and seleno-DL-methionine . The diets contained per kilogram the following aatioxidants : Diet 1, 0 mg vitamin E and 0 mg selenium ; Diet 2, 10 .5 mg vitamin E and 0 .04 mg selenium ; Diet 3, 45 mg vitamin E and 0 .20 mg selenium ; and Diet 4, 500 mg vitamin E and 0 .80 mg selenium . The animals were on a 14-hr dark and 10-hr light cycle with food provided only during the dark period . The rate were given deionized water ad libitum during the full 24-hr cycle . Phenobarbital-pretreated rats (four from each dietary group) were given 75 mg/kg doses of sodium phenobarbital iatraperitoneally on each of three days immediately prior to sacrifice . An equal number of rate received isotonic saline ae a placebo . All rats were starved for 24 hr prior to sacrifice . Rats were sacrificed in groups of two so that all determinations could be made in duplicate . After sacrifice by cervical dislocation, livers were excised, weighed, and homogenized in eight volumes of 50 _mM Tris-maleate buffer, pH 6 .5, that contained 155 _mM NaCl . Microsomes were isolated according to the procedure of Glende and Recknagel (18), and were suspended at a concentration of 1 ml per g of liver is the above buffer . The rate of NADPH-dependent microsomal lipid peroxidation was measured by oxygen uptake with a Clark-type oxygen electrode (Yellow Spriaga Instrument Co ., Yellow Springs, Ohio) . Typically, the reaction mixture contained 1 .4 ml of the Tris-maleate buffer, 1 .5 ml of microsome suspension, and 0 .1 ml of 18 mM NADPH . The buffer and microsomes were equilibrated to 37 ° C prior to addition of the NADPH to initiate lipid peroxidation . No oaygea uptake was observed prior to NADPH addition . When CC1 4 was required, 5 ul of CC1 4 was added to the buffer and soaicated for 30 sec prior to mixing with microsomes . The oxygen uptake was recorded continuously for the initial 5 min period following NADPH addition . With the usual addition of 1 .8 micromoles of NADPH, the oxygen uptake rate was linear for at least the first minute is all cases, and the initial velocity of the microsomal lipid peroxidation reaction was determined from this linear phase . Km estimation and NADPH/0 2 ration also were obtained from oxygen electrode experiments that used limiting concentrations of NADPH . Rm determinations were derived from reciprocal plots of substrate concentration versus initial velocity as obtained directly from the oxygen electrode measurements . Duplicate initial rate determinations were made at each substrate concentration . At least six separate substrate concentrations were utilized for each individual Km determination . The linear correlation coefficient for each Km determination was calculated . The NADPH/0 2 ratios were calculated from the amount of 0 2 utilized following the addition of known amounts of NADPH . Several different NADPH concentrations were utilized for each NADPH/0 2 ratio determination . NADPH/02 ration were not altered appreciably by changes is the NADPH concentration . 2 The basal diet contained 36X Torula yeast, 39X sucrose, 15X tocopheroletripped corn oil, SX salt mix as recommended by the National Research Council (17), except no selenium and the addition of 0 .05 g/kg sodium fluoride, and 5X vitamin mix in sucrose as recommended (17), except no vitamin E, and further additions of 1 ug/k8 biotin and 2 mg/kg folic acid .

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The extent of NADPA-dependent lipid peroxidation was measured by the malonaldehyde produced in 30 min at 37 °C . The reaction miaturea were identical The reaction was stopped after 30 with those used with the orygen electrode . min by the addition of trichloroacetic acid . Tha malonaldehyde was determined with thiobarbituric acid (Sigma Chemical Co ., St . Louis, Mo .) by the method of Wills (19) . Fatty acid analysis was performed by gas-liquid chromatography after a lipid extraction, according to the procedure of Bligh and Dyer (20), and preparation of fatty acid methyl asters by the technique of Metcalfe et al . (21) . Microsomal protein concentrations were determined by the method of Miller (22) . Cytochrome P-450 levels were measured with a Cary 118-C recording spectrophotometer from the carbon monoxide difference spectrum after reduction of both the refe~eacg and sample with dithionite . The molar extinction coefficient, 91 cm _mM , was used to quantitate cytochrome P-450 from the absorption difference between 450 and 490 nm (23) . NADPH-cytochrome ç reductase activity was measured by the initial rate of cytochrome ç reduction at 550 nm with the assay conditions of Masters et al . (24) . Results The rate and extent of _in vitro NADPH-dependent microsomal lipid peroxidation were affected considerably by dietary antioxidant levels and phenobarbital pretreatment . As shown in Table 1, the rate of lipid peroxidation, as measured by 02 uptake, and the extent of lipid peroxidation as measured by the amount of malonaldehyde that accumulated in 30 min, were both diminished as the amount of TABLE 1 Rate and Extent of Microsomal Lipid Peroxidation as a Function of Dietary Antioxidant, Phenobarbital, and CC14a Diet #

CC14

ul

02/min/mg protein

nmoles malonaldehyde/ 30 min/mg protein

without phenobarbital pretreatment 1 2 3 4

+ + + +

3 .5 3 .6 3 .2 3 .4 2 .6 3 .0 0 .8 2 .7

29 32 33 42 31 29 il 30

with phenobarbital pretreatment 1 2 3 4

+ + + +

3 .0 3 .1 3 .8 4 .2 2 .8 3 .2 3 .0 3 .7

aAll values are the average of duplicate determinations .

10 11 20 23 18 23 16 19

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The good correlation (r~0 .75) between 02 uptake dietary antioxidant increased . and malonaldehyde measurements in those experiments involving microsomes from animals not pretreated with phenobarbital shows that these two methods are equally appropriate for measurement of microsomal lipid peroaidation during the time periods selected . The data shown is Table 1 are the average of duplicate determinations on two groups of two animals each . Minor variations (

Effect of dietary antioxidants and phenobarbital pretreatment on microsomal lipid peroxidation and activation by carbon tetrachloride.

Life Sciences Vol . 19, pp " 1151-1160, Printed in the U .S .A . Pergamon Press 1976 . EFFECT OF DIETARY ANTIOXIDANTS AND PHENOBARBITAL PRETREATMEN...
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