Biochimica et Biophysics Acta, 1045 (1990) 99-106 Elsevier

BBALIP

99

53435

Lipid peroxidation in the liver of carcinogen-resistant

rats

Hanaa Hammad, Taneaki Higashi, Noriko Tateishi, Mitsuya Hanatani and Yukiya Sakamoto Division of Biochemistry, Department of Oncology, Biomedical Research Center, Osaka University Medical School, Fukushima, Osaka (Japan)

(Revised

Key words:

(Received manuscript

6 November 1989) received 27 February

Carcinogen-resistant;

Lipid peroxidation;

1990)

(Rat liver)

Recently, we developed a new strain of rats that exhibit marked resistance to the hepatotoxic and carcinogenic actions of 3’-methyl-4-dimethylaminoazobenzene (3’-MeDAB) and some other carcinogens. In this work, we compared lipid peroxidation in the liver of these carcinogen-resistant (R) rats and the parental Donryu strain rats that are sensitive (S) to hazardous actions of these carcinogens. The liver microsomal fractions of the R group contained less amounts of polyunsaturated fatty acids. Microsomal lipid peroxidation in the presence of exogenous NADPH was much lower in R rats than in S rats. Liver microsomes of R rats were much less active than those of S rats also in producing 4-hydroxynonenal, carbonyl compounds and conjugated diene. The hepatic contents of ascorbic acid, glutathione, a-tocopherol and coenzyme Q in the R rats were similar to those in S rats. The activities of the free radical scavenger enzymes, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), in the two groups were also similar. Alcohol dehydrogenase (ADI-I) and aldehyde dehydrogenase (ALDI-I) are both thought to function in disposal of these cytotoxic aldehydes. The liver microsomal and mitochondrial ALDH activities of the two groups were similar. The ADH activity of the liver cytosolic fraction of R rats was nearly twice that of S rats, as measured with 4-hydroxynonenal as substrate. The higher ADH activity may explain the decreased lipid peroxidation in R rats at least partly, if this enzyme is involved in lipid peroxidation.

Introduction Recently, we established a strain of rats that exhibits marked resistance to the hepato-toxic and carcinogenic actions of 3’-methyl-4-dimethylaminoazobenzene (3’MeDAB) as indicated by much lower y-glutamyltransferase activity and a low incidence of hepatoma after long-term exposure to the carcinogen. This strain was selected by exposure of carcinogen-sensitive (S) Donryu strain rats to 3’-MeDAB for more than 20 successive generations. These carcinogen-resistant (R) rats are also refractory to the actions of several other carcinogens

Abbreviations: MDA, malondialdehyde, TBARS, thiobarbituric acid-reactive substances; CHP, cumene hydroperoxide; t-BHP, tertiary butylhydroperoxide; HNE, Chydroxynonenal; PUFA, polyunsaturated fatty acids; 3’-MeDAB, 3’-methyl-4-dimethylaminoazobenzene; ALDH, aldehyde dehydrogenase; ADH, alcohol dehydrogenase; TBA, thiobarbituric acid. Correspondence: H. Hammad, Division of Biochemistry, Department of Oncology, Biomedical Research Center, Osaka University Medical School, l-l-50, Fukushima, Fukushima-ku, Osaka 553, Japan. 00052760/90/$03.50

0 1990 Elsevier Science Publishers

B.V. (Biomedical

including 3’-hydroxymethyl4-dimethylaminoazobenzene, 4-dimethylaminoazobenzene and 2-acetylaminofluorene [l-5]. The active metabolites of most carcinogens are thought to evoke formation of oxygen-derived free radicals such as superoxide (0;)and the hydroxyl radical (6H) and intermediate oxygen-reduction products such as hydrogen peroxide (H202) and their actions may result in ubiquitous cell damage through peroxidation of polyunsaturated fatty acids (PUFA) in membrane phospholipids and other cellular constituents [6,7]. Actually, clear parallelism has been found between the carcinogenicity of a given xenobiotic and the amount of active radicals generated and special attention has recently been paid to the possible role of these radicals in tumor initiation and promotion [8,9]. On the other hand, although lipid peroxidation seems to be a common devastating consequence of oxygen free-radicals and its role in producing structural and functional deformities of tissues has frequently been suggested [lO,ll], its exact mechanism(s) of action is (are) still obscure and ambiguous. The processes of lipid peroxidation in different cellular and subcellular systems and implications of their effects on various cellular Division)

100 functions have been reviewed [12,13]. There are several reports on the deleterious effects of lipid peroxidation, including enzyme inactivation, depletion of cofactors and the formation of degradation products, some of which are chemically reactive and mutagenic per se [14,15]. The rate of lipid peroxidation of subcellular fractions in vitro depends on several factors such as the presence of metal chelators, free-radical generating systems, pathways for metabolic activation and detoxification, the levels of cofactors such as NADPH, the concentrations of substrates such as PUFA and the activities of several types of radical-scavenging systems [1215]. Mechanisms have been proposed to explain diminished lipid peroxidation on the basis of reduction in NADPH-cytochrome P-450 electron transport and increase in lipid-soluble antioxidants often associated with reduction in the content of PUFA [16]. The present paper reports studies on the reaction sequences of lipid peroxidation and protective and detoxification systems in the liver of S and R strain rats to shed some light on the reason(s) for the increased resistance of R rats to oxidative hazard and their better protection from the deleterious effect of lipid peroxidation. Materials and Methods Chemicals Thiobarbituric acid (TBA), a-tocopherol, standard fatty acids (palmitic, stearic, oleic, linoleic, linolenic and arachidonic), ubiquinone-10 (UQ,,,) and ubiquinone-9 (UQs) from bovine heart, glutathione reductase (EC 1.6.4.2) and isocitrate dehydrogenase (EC 1.1.1.42) were provided by Nacalai Tesque (Kyoto, Japan). Cytochrome c (horse-heart, type III), xanthine oxidase (EC 1.2.3.2) from butter milk, grade I and superoxide dismutase (EC 1.15.1.1) from bovine erythrocytes were from Sigma (St. Louis, MO, U.S.A.). Ultra-filtration membranes were from Amicon (Donvers, MA). 4-Hydroxynonenal was a generous gift from Dr. Mitsuyoshi Matsuo (Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan). Other chemicals used were of the highest purity available commercially. Animals and treatment Carcinogen-sensitive Donryu rats were supplied by Nippon Rat Co. (Saitama, Japan) and maintained for at least 1 week in our laboratory before use. The carcinogen-resistant strain rats were obtained as described in detail previously [1,2]. Carcinogen-resistant rats were maintained on carcinogen-free basal diet (commercial laboratory chow, MF; Oriental Yeast, Tokyo Japan) for at least three generations before experiments, to avoid the influence of exposure to 3’-MeDAB and its metabolites through the uterus, milk, or food. Male S and R rats of 8-9 weeks old weighing 250-300 g were divided into two groups. One group was kept on basal

diet and the other 0.06% 3’-MeDAB were housed in an controlled lighting diet were available

group was given a diet containing for the indicated periods. All rats air conditioned room at 22°C with (12-h light-dark cycle). Water and ad libitum.

Liver tissue preparation Rats were killed by decapitation under light ether anesthesia and the liver was quickly excised, chilled in ice-cold 1.15% KCl, rinsed several times to remove blood, then dried on filter paper and weighed. All subsequent procedures were carried out at 0-4OC. Pieces of liver were taken at random from different lobes and homogenized in 10 ~01s. of 0.05 M potassium phosphate buffer (pH 7.0) for catalase assay, or in 4 ~01s. of 0.05 M Tris-maleate buffer (pH 6.8) for other experiments in a Potter-Elvehjem Teflon-glass homogenizer. Subcellular fractions (the postnuclear supernatant, mitochondria, microsomes and cytosol) were prepared by differential centrifugation. Samples were frozen at - 70 “C until use. Lipid peroxidation was assayed within 24 h and enzyme analyses within 72 h. Lipid peroxidation. Lipid peroxidation reactions were driven enzymatically as follows: (a) incubating the microsomal suspension (approx. 1.0 mg/ml 0.15 M KCl) with 0.1 M phosphate buffer (pH 7.3) and NADPH (160 PM final concentration) at 37°C under air with constant shaking for up to 60 min and samples of 1 ml of the mixture were removed at interval for assay of MDA. Cumene hydroperoxide was used at a final concentration of 1 mM in place of NADPH to assess NADPH-independent rnicrosomal lipid peroxidation in the liver of S and R rats (for Fig. 1); (b) a complete incubation mixture consisted of (in a final concentration) microsomal suspension from 50 mg of liver/ml 0.15 M KCl, 0.05 M Tris-maleate buffer (pH 6.6) 0.025 mM FeSO,, 0.04 mM NADP+, 5 mM MgCl,, 2.5 mM ADP, 20 mM Dr_-isocitrate and isocitrate dehydrogenase at 10 mm-&s/ml. The incubation was carried out aerobically for 60 min at 37 o C in an Erlenmeyer flask with constant shaking. At the end of the incubation period, 15 PM EDTA was added to the mixture to prevent further peroxidation (for all other experiments except Fig. 1). MDA. MDA was measured by method of Ohkawa et al. [17]. For some experiments, free and bound MDA were measured directly by HPLC (as described in Ref. 18). Diene conjugate. Diene conjugate was determined by the method of Recknagel and Ghoshal [19] as modified by Lee et al. [20]. 4-Hydroxynonenal. 4-Hydroxynonenal was separated and quantitated by the method of Esterbauer [21]. Fatty acid analysis. Major fatty acids of microsomal total lipid were analyzed quantitatively by gas chromatography as described by Jordan and Schenkman

101 Glutathione peroxidase [EC 1.11.1.91 (GSH-PX) was measured as described by Mohandas et al. [28]. Phospholipid hydroperoxide-glutathione peroxidase activity was assayed as described by Paglia and Valentine [29] but with a modification of the reaction mixture [301-

Alcohol dehydrogenase [EC 1.1.1.11. Cytosolic (ADH) was assayed as described by Btittner [31]. Aldehyde dehydrogenase [EC 1.2.1.31. Microsomal and mitochondrial (ALDH) was assayed as described by Tottmer [32].

e-0

+-a ’

60

Antioxidant assay

Minutes

Fig. 1. Lipid peroxidation in rat liver microsomes of S and R rats. Production of thiobarbituric acid-reactive substances (TBARS) by liver microsomes of S and R rats was measured during aerobic incubation of microsomes (approx. 1 mg protein/ml) with 160 PM NADPH or with 1 mM cumene hydroperoxide at 37’C with constant shaking for the time indicated in the figure. Experimental conditions are given in the text. 0, 0, a, results for 14 S rats under control, in presence of NADPH, or CPH, respectively; 0, n, rats under control, in presence of NADPH and peroxide, respectively. Mean va1uesfS.D. of 14 tions for S and R rats, respectively, are

0, results of 11 R or cumene hydroand 11 determinashown.

[22] after lipid extraction as described previously [23]. Fatty acid methyl ester were prepared as described in Ref. 24. Enzyme assays

NADPH-cytochrome c reduction [EC 1.6.2.41 was assayed as described previously [25]. Catalase [EC 1.11.1.61 (CAT) activity was measured by the method of Cohen [26]. Superoxide dismutase [EC 1.15.1.11 (SOD) activity was measured by the method of Beyer and Fridovich ~271. TABLE

Freshly prepared liver homogenates were used for estimating the contents of vitamin C (ascorbic acid) as described by Omaye et al. [33]. Liver microsomal vitamin E (a-tocopherol), ubiquinones 9,lO and their reduced forms ubiquinols 9,lO were determined as previously described [34,35]. Protein determination

Protein concentration was determined by the method of Lowry et al. [36] with bovine serum albumin as a standard. Statistical analysis

Results are expressed as means f S.D. Student’s t-test was used to determine the significance of differences between values for different experimental groups. Results Production (TBARS)

of

thiobarbituric

acid-reactive

substances

Formation of TBARS is widely used as an index of the extent of lipid peroxidation. Fig. 1 shows that liver

I

Time-dependence of formations of conjugated diene, carbonyl compounds and I-hydroxynonenal carcinogen-resistant (R) rats

by liver microsomes of carcinogen-sensitive (S) and

Suspensions of liver microsomes of S and R rats were incubated aerobically with an ADP/Fe 2+ . NADPH-generating system for 10 and 60 mm at 37 o C with shaking. Then microsomal lipids were extracted and the contents of conjugated diene, carbonyl compounds and 4-hydroxynonenal were measured. Values are means* S.D. for groups of 14 rats. * and * * indicate a significant difference from the corresponding value for S rats at P < 0.05 and P < 0.02, respectively; n.d., not detected. Rat

S

ADP/Fe 2+ : NADPHgenerating system

+ +

R

+ +

Incubation time

(nmol/mg

protein)

(mm)

conjugated diene

carbonyl compounds

4-hydroxynonenal

10 60 10 60

11.6+ 0.5 12.3* 1.3 69.9* 8.3 98.4 f 20.1

82.0 f 89.1 f 226.3 f 273X*

n.d. n.d. 5.4* 1.8 8.9*1.1*

10 60 10 60

9.3f 3.2 12.2* 1.7 56.9 f 10.6 66.7 f 17.2

55.3* 6.9 59.0* 14.1 158.0 f 25.2 191.0* 14.1* *

14.0 12.0 36.1 21.9 * *

n.d. n.d. 4.7 f 0.3 5.3 f 1.0 *

102 TABLE

Ha

TABLE

Fatty acids conients of liver of S and R rats Values are means + S.D. for groups (nmol/mg

Fatty acids

C,,:o C,s:o+,s., Cl,:2

C,,:, c20:4

not detected.

R f 25.2 f 34.1 f 18.1 f 38.8

Ejjecf of a lipid peroxidation distributions in S and R rats

214.3 f 10.9 225.8 f46.9 135.4 f 36.8 n.d. 179.6k31.3

Fatty acids

Control S

microsomal suspensions from R rats produced much less TBARS than those from S rats when incubated aerobically with exogenous NADPH. Measurement of change in TBARS with time during incubation at 37 o C showed that there was no remarkable difference in TBARS production by microsomes of S and R rats in the first 5 min after addition of a final concentration of 160 PM NADPH, but after 30 rnin, TBARS production by microsomes of S rats was nearly 2-fold higher than that of microsomes of R rats and this difference between the groups did not change during incubation till 60 min. The TBARS production by the R group during lipid peroxidation induced by cumene hydroperoxide was much greater and so the difference in TBARS productions in the two groups was not clearly observed. The rate of TBARS production with cumene hydroperoxide was rapid in the first 5 min and then the rate declined during the rest of the incubation period examined in both groups. Isolated liver microsomes of both strains of rats produced low but comparable amounts of TBARS during incubation at 37” C in the absence of any factor stimulating lipid peroxidation. Formation of intermediates of lipid peroxidation The extent of peroxidative decomposition of rat liver microsomal structural lipid was assayed quantitatively by measurements of diene conjugate, carbonyl compounds and hydroperoxide degradation aldehydic products, e.g., 4-hydroxynonenal. As shown in Table I, under basal conditions, no 4-hydroxynonenal was detected in the preparations from either S or R rats, but carbonyl compounds were detectable at slightly, but not signifi-

TABLE

generating

system

on the fatty

acids

Freshly prepared liver microsomes of S and R rats were incubated without (control) and with an ADP/Fe’+ : NADPH-generating system at 37 o C for 60 min. Their fatty acid contents were then analyzed by capillary GC as described in the Materials and Methods. Values shown are peak area 48 in fractions (n = 6). Values are means f S.D. for percentage contents (peak areas) in the total lipid fraction in groups of six rats; n.d.. not detected.

protein)

S 189.7 226.3 132.6 n.d. 249.3

of four rats, nd.,

IIb

C,s:o+,s:, C,,:, C,,:, C 20.4

R

33.3 f 3.2 14.4* 1.7 n.d. 18.0f 1.9

34.8 f 1.4 15.4*0.7 nd. 15.6 & 2.2

ADP/Fe 2+ : NADPHgenerating system S

R

30.7 f 2.2 9.9+2.1 n.d. 5.3*1.4

33.0 * 2.3 8.3 + 2.3 nd. 5.0*0.7

cantly, different levels in the two groups. The basal levels of conjugated diene in S and R rats were similar. When microsomal lipid peroxidation was stimulated by adding ADP/ Fe’+ and an NADPH generating system, the productions of conjugated diene and carbonyl compounds increased markedly and time-dependently in both S and R rats. However, the formation of conjugated diene and carbonyl compounds by microsomes from R rats were significantly lower, especially after 60-min incubation with the lipid peroxidation system (Table I). The production of 4-hydroxynonenal by microsomes of S and R rats during incubation with the lipid peroxidation system was similar during the first 10 min, but after 60 min the production by microsomes of R rats was only half that of microsomes of S rats. Consistent with results on production of TBARS after stimulation of lipid peroxidation by exogenous NADPH (Fig. l), these results indicate that stimulated formation of lipid peroxidation products in microsomes from R rats decreased prematurely. Microsomal PUFA contents and dissociation of PUFA during NADPH-Fe2 +-dependent lipid peroxidation The contents of some major fatty acids as stearic, oleic and linoleic acids in the microsomal fractions of S and R rats were nearly similar. The linolenic acid con-

III

Antioxidant

contents of the liver of carcinogen S and R rats

This table shows the contents forms [ubiquinol-9 (UQ9H2) shown in parentheses. Rat

Ascorbic

S R

1327*133(5) 1340 f 154 (6)

acid

of ascorbic acid, a-tocopherol, and ubiquinol-10 (UQ,0H2)].

oxidized ubiquinones [ubiquinone-9 (UQs), ubiquinone-10 (UQro)] and their reduced Values are given in nmol/g liver tissue and are means& SD. for the numbers of rats

a-Tocopherol

UQ,H,

UQIOH,

UQ9

UQto

41.5 k6.0 (8) 50 *8.2 (4)

175 f 29 (4) 150* 19 (5)

18.3 + 1.6 (3) 12.7 * 1.5 (5)

5.56 f 1.16 (5) 6.35 50.77 (3)

1.45 f 0.07 (5) 1.05 f 0.05 (4)

103 tents were too low to be measured accurately. The arachidonic acid content was detectably lower in the R group than in the S group (Table IIa). We found that incubation of the microsomal fractions of S and R rats with ADP/Fe’+ and an NADPH-generating system for 60 min at 37’ C resulted in no detectable change in the percentage contents of stearic and oleic acids, whereas the linoleic acid and arachidonic acid content decreased to about 50 and 40% of the initial values, respectively, in both strains of rats (Table IIb). Inhibitors of lipidperoxidation in the liver of S and R rats

Table III summarizes results on the concentrations of ascorbic acid, a-tocopherol and ubiquinol-ubiquinone 9,lO in liver homogenates of S and R rats. Their concentrations were not significantly higher in R rats than in S rats. We found previously that the glutathione concentrations in S and R rats were not significantly different [3]. Effect of diet containing 3’-MeDAB on enzymes related to lipid peroxidation process NADPH-cytochrome c reductase activity and NADP(H) content. There is evidence that NADPH-cyto-

chrome c reductase activity is involved in promoting NADPH-dependent lipid peroxidation [25]. However, we found no detectable difference in the reductase activities of microsomal suspension from S and R rats red on basal diet (Table IV). Some xenobiotics that undergo NADPH-linked transformation are known to inhibit lipid peroxidation in liver [37]. But, we found that administration of diet containing 3’-MeDAB for 1 week did not decrease NADPH-cytochrome c activity appreciably. We found that the concentrations of NADPH and NADP+ in the liver of R rats and S rats were similar (data not shown). Also, we have noted that TABLE

TABLE

IV

Effect of diet containing 0.06 % 3’ -methyl-4-dimethylaminoazobenrene (3’-MeDAB) for I week on the activity of NADPH-cytochrome c reductase in liver microsomes of S and R rats Values are means + S.D. for five and six determinations rats, respectively. Diet

Rats

NADPH-cytochrome c reductase (nmol product formed/ mm per mg protein)

Basal

S R

107.8 + 9.6 113.8k5.6

3’-MeDAB

S R

101.6 f 7.6 94.6k7.9

the total cytochrome P-450 content of liver microsomes of two strains are similar [l]. Activities of oxygen-scavenging enzymes. Table V shows that unexpectedly, the R group did not show higher activities of any of the scavenging enzymes, superoxide dismutase (Cu, Zn SOD, Mn SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) than the S group. We found that administration of diet containing 3’-MeDAB for 1 week caused a similar marked increase in the activity of liver catalase in the S and R rats, but that it did not affect GSH-Px or SOD activity appreciably (Table V). Microsomal GSH-Px activity was comparable in both S and R rats (data not shown). Detoxification of cytotoxic aldehydic products: possible roles of aldehyde and alcohol dehydrogenases. The NAD-

dependent aldehyde dehydrogenase (ALDH) activities in the liver microsomal and mitochondrial fractions of S and R rats are shown in Table VI. The subcellular distribution of enzyme activity was similar in the two groups with acetaldehyde, 4-hydroxynonenal or MDA as substrate. The enzyme showed lower activity with

V

Effects of 3’-MeDAB

diet on activities of scavenging enzymes of active oxygen species in S and R rats

Rats were given basal diet containing 0.06% 3’-MeDAB for 1 week. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) (CAT) in the liver were measured as described previously. Values are means f S.D. for the numbers of rats shown in parentheses. Diet

Basal

3-MeDAB

for S and R

Rats

SOD a

GSH-Px

Cu-Zn

Mn

S

63.1 f 5.6

R

(5) 74.3f7.3

b

and catalase

CAT =

cumene hydroperoxide

t-BHP

Hz02

PLOOH

40.3 It4.5

1.20*0.30

0.83 kO.31

0.81 f0.30

0.046 f 0.009

343.90f

(3) 39.0&-2.0

(14) 1.30f0.30

(14) 0.92 f 0.24

(14) 0.88 f 0.26

(4) 0.032 f 0.001

(8) 349.20 f 53.20

36.30

(6)

(3)

(14)

(14)

(14)

(4)

(8)

S

72.3 f 8.8

41.Ok6.3

1.24*0.10

0.74 f 0.02

0.69f0.15

0.037 *0.005

505.90 f 82.10

R

(4) 70.6k2.1

(4) 31.3k2.2

(4) 1.40*0.10

(4) 0.81 f 0.30

(4) 0.76 f 0.08

(4) 0.035 * 0.003

(4) 464.75 f 90.50

(4)

(4)

(4)

(4)

(4)

(4)

(4)

a 1 unit of SOD is defined as that causing 50% inhibition of cytochrome b amol HADPH oxidized/mitt per mg protein (cytosol). ’ mm01 H,O* decomposed/mm per mg protein (postnuclear supematant).

c reduction

(cytosol

and mitochondtia).

104 TABLE

VI

Intracellular distribution of aldehyde dehydrogenase (ALDH) (nmol NADH formed/m&

per mg protein) in livers of S and R rats

Enzyme activity was assayed as described in the Materials and Methods. The reaction was started by adding acetaldehyde, 4-hydroxynonenal or malondialdehyde as substrate. Sodium deoxycholate (0.25 mg/mg of protein sample) was used to release the latent activity of ALDH in the mitochondrial fraction. Rats were given basal diet or diet containing 0.06% 3’-MeDAB for 1 week. Results are means+ SD. for the numbers of rats shown in parentheses. nd., not detected. Subcellular fractions

Rats

ALDH acetaldehyde basal

Microsomes

Mitochondria

4-hydroxynonenal 3’-MeDAB

malondialdehyde

basal

3’-MeDAB

basal

3’-MeDAB

9.2k4.4

7.2

35.9 + 1.5

11.6k5.6

n.d.

n.d.

5.3

(7) 44.2 f 4.5

(11) 12.5 f 4.9

(5) 15.4k2.1

6.8f1.3

nd.

(7)

(7)

(12)

(4)

(4)

S

31.8+10.3

27.7 f 4.3

5.2+0.8

R

(11) 29.5 f

(6) 29.5 f 5.9

(6) 5.9+ 1.6

(7)

(7)

S

31.2*

R

(9) 37.85

7.5

(11)

4-hydroxynonenal or MDA than with acetaldehyde, although the relative activities of the mitochondrial enzymes with these substrates were somewhat different (Table VI). Administration of a diet containing 3’MeDAB for 1 week did not have a marked effect on the enzyme activity in either S or R rats, although R rats showed slightly higher enzyme activities, particularly in their microsome fraction, after this treatment. Reduction of toxic aldehydes to the corresponding less toxic alcohols by liver alcohol dehydrogenase (ADH) is suggested- to be a main route of intracellular detoxification [38]. With either basal or carcinogenic diet, cytosolic ADH activity in the liver was found to be nearly twice as high in R rats as in S rats with either

-

n.d. 5.4*0.3 (5)

9.2 + 3.6

8.1+ 1.9

(9) 7.5 f 3.6

(4) 6.351.8

(12)

(7)

acetaldehyde or 4-hydroxynonenal as substrate (Fig. 2). Administration of 3’-MeDAB diet reduced the enzyme activity in both S and R rats although the activity was still higher in the latter. From the data in Table VII we noticed that the addition of cytosol to the microsomes mixture caused relative decreasing in the production of TBARS in S and R rats. This decreasing became significant when the cytosolic fraction of R rats was added to the microsomes of S rats, while adding the cytosol of S rats to the microsomes of R rats had nearly no effect on the production of TBARS. To give assurance that cytosolic ADH has a main role in decreasing TBARS, we added pyrazole (specific inhibitor of ADH) to the mixture of microsomes and TABLE

VII

Effect of pyrazole on lipid peroxidation (TBARS-production) Microsomes

Malondialdehyde

4-Hydroxynonenal

Acetaldehyde

Fig. 2. Activities of cytosolic alcohol dehydrogenase (ADH) in the liver of S and R rats measured with various substrates and effects of 3’-MeDAB on the activities. Concentrations of 15 mM malondialdehyde, 0.5 mM 4-hydroxynonenal and 5 mM acetaldehyde was used as substrates. Activity was measured spectrophotometrically as described in Materials and Methods. Cl, q, values for 14 S and 17 R rats respectively, maintained on control diet; q, n, values for 12 S and 7 R rats, respectively, maintained on diet containing 0.06% 3’-MeDAB for 1 week. * P < 0.05; * * P -C0.025.

a

Cytosol b

S S S S S

S S R R

R R R R R

R R S S

_

Pyrazole ’

TBARS d

_ _

7.1*1.1 5.7f0.9 6.7kl.O 3.3 f 0.4 * 5.3f0.5 *

+ _ + _ _ + +

3.2 f 0.3 * * 2.4* 0.1 * * 2.8 f 0.7 3.3 f 0.4 3.8f0.3

L See lipid peroxidation reaction mixture. 0.1 ml crude cytosol fraction was added to (a) before incubation. ’ 0.01 ml of 30 mM pyrazole was added to the mixture (a + b) before incubation. d Values are means + S.D. for four determinations for S and R rats expressed as nmol/mg protein. * P