EFFECT OF VITAMIN C DEFICIENCY ON THE METABOLISM OF DRUGS AND NADPH-LINKED ELECTRON TRANSPORT SYSTEM IN LIVER MICROSOMES

EFFECT

OF

BOLISM

OF

VITAMIN

C DEFICIENCY

DRUGS

TRANSPORT

AND

SYSTEM

RYUICHI

NADPH-LINKED IN

KATO,*

ON

LIVER

AKIRA

THE

META

ELECTRON

MICROSOMES

TAKANAKA*

AND TAKAO OSHIMA** *Departmentof Pharmacology , NationalInstituteof HygienicSciences,Setagapa-ku,Tokyo "Department of Pharmacology , Universityof Keio,Schoolof Medicine,Shinjuku-ku,Tokyo Received for publication July 15, 1968

Various drugs are inactivated by liver microsomes in the presence of NADPH and atomospheric oxygen (1). The enzymes catalyzing these reactions can activate molecular oxygen by a two-electron reduction so that one oxygen atom is introduced into the substrate leading to a hydroxylated product, whereas the second atom is reduced to water (1, 2). Recently the participation in these reactions of hemoprotein called cytochrome P-450 (3) as the oxygen-activating component (4-8) has been established (Fig. 1).

FIG. 1. Schematic

representation

oxidizing systems fp : Flauoprotein,

of

NADPH-linked

of liver microsomes. X : Unknown factor,

electron

transport

NT : Neotetrazolium,

and

drug

RH : Substrate

(Drugs)

The activity of drug-metabolizing enzymes of liver microsomes was altered by various factors, such as the administration of phenobarbital or methylcholanthrene (9, 10), thy roxine (11-13), anabolic hormone (11, 14, 15), carbon tetrachloride (11, 16) and morphine (11, 12), and adrenalectomy (11, 17), thyroidectomy (13), hepatectomy (18), starvation (19, 20), alloxan diabetes (11, 12) and low protein diet (16, 21, 22). It was also demonstrated that the activity of NADPH-linked electron transport system of liver microsomes was often altered in association with the alteration in the activity of drug-metabolizing enzymes under the above-given conditions (10, 13, 22, 26). Vitamin C is a well known component related to the control of oxido-reduction states of living cell, but detailed role of vitamin C has not been fully elucidated (27) . On Preliminary communication was reported at 39th Annual Society in Nov. 1966 (Seikagaku 36, 355, 1966).

Meeting

of the Japanese

Biochemical

the other hand, Mitoma et al. (28), Tochino et al. (29) and more recently, Conney et al. (30) reported that the hydroxylation of acetanilide, hexobarbital and zoxazolamine was decreased in vitamin C deficient guinea-pigs. However, the studies on the mechanism of decreased hydroxylation activity in relation to the activity of NADPH-linked electron transport system has not yet been reported. The purpose of the present study, therefore, is to investigate whether or not the mechanism of decreased hydroxylation activity of liver microsomes from vitamin C deficient guinea-pigs is related to the decreased activity of NADPH-linked electron transport system. MATERIALSAND METHODS Male guinea-pigs, weighing about 340-440 g were used. Five to six animals were used for the one experiment. The animals were fed on a vitamine C free synthetic diet for 12 days before sacrifice. The percentage composition of the vitamin C free synthetic diet was as follows : Vitamin-free casein, 30; corn starch, 20; glucose, 10.6; sucrose, 10; wood pulp, 10; agar, 15; salts, 6 ; cottonseed oil, 5 ; potassium acetate, 2.5; magnesium oxide, 0.5; inositol, 0.2 and choline chloride, 0.2. Vitamins were added in amounts of provide for each kilogram of diet the following: (in milligrams) thiamine HC1, 16; riboflavin, 16; pyrido xine HC1, 16; Ca pantothenate, 40; nicotinic acid, 200; biotin, 1; folic acid, 10; 2 methyl-naphthoqui none, 5; p-aminobenzoic acid, 100; ce-tocopherol, 100; vitamin B12, 50; also vitamin A, 6000IU and vitamin D, 600 1U. The control animals were daily treated with ascorbic acid (20 mg/kg, i.p.). The animals were killed 24 hours after the last injection. Preparation of liver microsomes: The animals were decapitated and the livers were removed, chopped into small pieces, washed well, and homogenized with 3 volumes of 1.15% (isotonic) KC1 solution in a Teflon-glass homogenizer. The homogenates were centrifuged at 9,000 x g for 20 minutes. The supernatant solution were then centrifuged at 105,000 x g for one hour, and the microsomes were suspended in 1.15% KCI solution. Assays of drug-metabolizingactivities: The incubation mixtures consisted of 9,000 x g supernatants equivalent to 625 mg of liver, 20 ,moles of glucose-6-phosphate, 0.6 ,umoles of NADP, 50 moles of nicotinamide, 50 moles of MgC12j 1.4 ml of 0.2 M sodium phos phate buffer (pH 7.4), various substrates, and water to a final volume of 5.0 ml. The following amounts of the substrates were used: hexobarbital, 3.0 ,umoles; aminopyrine, 5.0 ,moles; aniline, 5.0 ,umoles; zoxazolamine, 1.0 ,umoles; p-nitrobenzoic acid, 5.0 ,umoles; p-dimethylaminoazobenzene, 2.0 ,umoles; N-methylaniline, 5.0,umoles; diphenhydramine, 5.0 ,umoles; meperidine, 5.0 p moles; p-nitroanisole, 5.0 ,umoles. The mixtures were incubated at 37°C for 30 minutes under air except p-nitrobenzoic acid and p-dimethylaminoazobenzene which were incubated in an atmosphere of nitrogen. The hydroxylation of hexobarbital and zoxazolamine was determined by measuring the disappearance of the substrates according to the method of Cooper and Brodie, and

Conney et al., respectively (31, 32). The hydroxylation of aniline was determined by measuring the formation of p-amino phenol according to the method described in a previous paper (20). The N-demethylation of aminopyrine was determined by measuring the formation of 4-aminoantipyrine according to the method of La Du et al. (33). The N-demethylation of N-methylaniline, diphenhydramine and meperidine was determined by measuring the formation of formaldehyde according to the method of Nash (34). The O-demethylation of p-nitroanisole was determined by measuring the formation of p-nitrophenol (20). The nitro-reduction of p-nitrobenzoic acid was determined by measuring the formation of p aminobenzoic acid according to the method of Fouts and Brodie (35). The azo-reduction of p-dimethylaminoazobenzene was determined by measuring the disappearance of the substrates according to the method of Conney et al. (36). The all activities were expressed as mtemoles of substrate disappeared or metabolite formed by gram wet weight of liver. Assaysof activitiesof electrontransportsystemsin liver microsomes: The activity of NADPH cytochrome c reductase was determined according to the method of Williams and Kamin (37). The activity of NADPH-neotetrazolium reductase was determined according to the method described in a previous paper (26). The activity of NADH-cytochrome c reductase was determined by the method used for NADPH-cytochrome c reductase. The activity of NADH-ferricyanide reductase was determined according to the method of Wil liams and Kamin (37). The activity of NADPH oxidase was assayed spectrophotometri cally according to the method of Gillette et al. (1). The activity of NADH oxidase was determined by the method used for NADPH oxidase. The content of cytochrome P-450 and b, was determined as described in a previous paper (13). Determinationsof microsomalprotein and liver and kidneyascorbicacid: Hepatic microsomal protein was measured according to the method of Lowry et al. (38). Liver and kidney (0.5 gram) were homogenized with 9.5 ml of 6% trichloracetic acid and the filtrates were used for the assays of ascorbic acid according to the method of Roe (39). RESULTS

1. Effect of vitamin C deficiencyon body weight, liver weight, liver microsomalprotein liver and kidney ascorbic acid content The body weight and liver weight of the guinea-pigs fed vitamin C deficient diet were not significantly altered and the protein content of the liver microsomes was also not sig nificantly altered (Table 1). The ascorbic acid contents of the liver and kidney of the guinea-pigs fed vitamin C deficient diet were markedly low and about 9% of control. 2. Effect of vitamin C deficiencyon the hydroxylationof aniline, hexobarbital and zoxazolamine by liver microsomes The hydroxylation of aniline, hexobarbital and zoxazolamine was markedly decreas ed in the guinea-pigs fed vitamin C free diet, and these results were in accordance with Axelrod et al. (28), Tochino et al. (29), and Conney et al. (30) (Table 2).

TABLE 1.

Effect

of

microsomal

vitamin protein

C and

deficiency liver

on

and

body

kidney

weight,

ascorbic

liver acid

weight,

liver

content.

All guinea-pigs were fed vitamin C free diet for 12 days and the control was daily treated with ascorbic acid (20 mg/kg, i.p.) for 11 days. The animals were sacrificed 24 hours after the last injection. The results are expressed as averages± S.E. The figures in parentheses indicate the number of animals used. TABLE 2.

Effect

barbital

See

the

legends

of and

for

vitamin

C deficiency

zoxazolamine

Table

by

on liver

the

hydroxylation

of

aniline,

hexo

microsomes.

1.

It was likely that there was no clear difference in the magnitude of the decrease amoung the hydroxylation of aniline, hexobarbital and zoxazolamine. 3. Effect of vitamin C deficiencyon the N-demethylationof aminopyrine,diphenhydramine , N methylanilineand meperidineby liver microsomes In contrast to the hydroxylation of drugs, the N-demethylation of aminopyrine was not significantly decreased (Table 3). Therefore, the effect of vitamin C deficiency was investigated with the N-demethylation of other substrates , such as diphenhydramine, N methylaniline and meperidine. However , the N-demethylation of all the substrate was TABLE 3.

Effect

of vitamin

diphenhydramine,

See

the

legends

for

C deficiency N-methylaniline

Table7~1.

on

the and

N-demethylation meperidine

of aminopyrine by

liver

microsomes.

,

not significantly altered as well as the N-demethylation of aminopyrine. These results indicate that the activity of N-demethylation of drugs by liver microsomes was not signi ficantly influenced by vitamin C deficiency. Since the alteration of aminopyrine N-de methylation has been almost parallel to that of hexobarbital hydroxylation under unphy siolosical conditions (12, 13, 20-26), the results of the present investigation show a special interest for the specificity of drug-metabolizing enzymes. 4. Effect of vitamin C deficiencyon the 0-demethylationof p-nitroanisole and the reduction of p nitrobenzoicacid and p-dimethylaminoazobenzene by liver microsomes Similarly, the activity of O-demethylation of p-nitroanisole was not significantly altered by vitamin C deficiency (Table 4). Moreover, the nitro-reduction of p-nitrobenzoic aicd and the azo-reduction ofp-dimethylaminoazobenzene were not significantly altered. These results indicates that the effect of vitamin C deficiency on the microsomal drug-metaboliz ing enzymes is likely specific and the hydroxylating reaction is only significantly affected. TABLE 4. Effect

See

the

of

vitamin

anisole,

the

benzene

by liver

legends

for

C deficiency

reduction

Table

on

of p-nitrobenzoic

the

O-demethylation

acid

and

of p-nitro

p-dimethylaminoazo

microsomes.

1.

TABLE5. Effect of vitamin C deficiency on the activity of NADPH-cyt c reductase, NADPH-neotetrazolium (NT) reductase, NADH-cyt c reductase and NADH-ferricyanide reductase of liver microsomes.

5. Effect of vitamin C deficiencyon the activitiesof NADPH and NADH-linked electrontrans port systemsin liver microsomes The activity of NADPH-cytochrome C reductase, NADPH-neotetrazolium reductase, NADH-cytochrome c reductase and NADH-ferricyanide reductase was not altered in the guinea-pigs fed vitamin C deficient diet (Table 5) and the activity of NADPH oxidase and NADH oxidase was also not altered. Moreover, the content of cytochrome P-450 and b, was not altered in vitamin C deficiency (Table 6). These results indicate that the

TABLE 6.

Effect

NADH

See

the

legends

of

vitamin

C

oxidase

and

the

for

Table

1.

deficiency content

of

on

the

activity

cyt

P-450

and

of b5

NADPH

in liver

oxidase,

microsomes.

decrease in the hydroxylating reaction of drugs by liver microsomes in the scorbutic animals is not mediated through the alteration in the activities of NADPH and NADH-linked electron transport systems. 6.

Time coursestudy on the effect of vitamin C deficiencyon the activity of drug-metabolizing enzymes of liver microsomes In further experiment, the alteration in the activity of the drug-metabolizing enzymes was investigated in the guinea-pigs fed vitamine C free diet for 6 days or 12 days (Fig. 2). In this experiment, the ascorbic acid content in the liver of 6 days deficient guinea-pigs was already 15% of control animals and the activity of zoxazolamine hydroxylation was 74% of control, whereas the activity of aniline hydroxylation was 79% (no significance) of control. After the 12 days deficiency the activity of zoxazolamine hydroxylation was

FiG. 2. Time course study on the effect of vitamin C deficiency on the activity of drug-metabolizing enzymes of liver microsomes. All the guinea-pigs fed vitamin C free diet for 12 days and the control was daily treated with ascorbic acid (20 mg/kg, i.p.) for 11 days, while the other group of animals was given ascorbic acid for 6 days (6 days deficient group). The animals were sacrificed 24 hours or 6 days after the last injection. The results were expressed as relative value (control= 100) obtained from each 5 guinea-pigs.

almost same to that of the 6 days deficiency, in contrast, the activity of aniline hydroxyla tion was progressively decreased and after the 12 days deficiency the activity was 52% of control. On the other hand, the activity of meperidine N-demethylation was 73% (no signi ficance) of control after the 6 days deficiency, but it was 102% of control after the 12 days deficiency. Similarly, the activity of diphenhydramine was 70% (p<0.05) of control after the 6 days deficiency, but it was 85% (no significance) of control after the 12 days deficiency. These results suggested a possibility that the activity of N-demethylase was may slightly decreased at the early stage of vitamin C deficiency, but after then the activity shows a tendency to return to control level. DISCUSSION Axelrod et al. reported that the activity of acetanilide and aniline hydroxylation by liver microsomes was markedly decreased in vitamin C deficient guinea-pigs (28). Later Tochino et al. reported that the hydroxylation of hexobarbital was decreased in vitamin C deficient guinea-pigs (29). Moreover, Conney et al. observed a marked decrease in the hydroxylation of zoxazol amine (30). These results on the hydroxylating activity were confirmed in the present investigation, whereas the N-demethylation of aminopyrine, diphenhydramine, meperi dine and N-methylaniline and the O-demethylation of p-nitroanisole was probably not significantly decreased. These results indicates that the N-demethylating activity is much less sensitive than the hydroxylating activity to vitamin C deficiency. However, the pos sibility of a decrease in the N-demethylating activity in more prolonged and heavy scor butic animals is not excluded from the present study. The discrepancy in the alteration of the activity of aminopyrine N-demethylation and hexobarbital hydroxylation are of special interest, because the both activities latered parallel under various abnormal condition (11-13, 20-26). These results also suggested a possibility that the hexobarbital hydroxylation and the aminopyrine N-demethylation were mediated through different enzymes. These results indicate that the mechanism of the decrease in the hydroxylation of drugs by liver microsomes in the vitamin C deficiency is not related to the activity of NADPH linked electron transport system, but it is probably related to the activity of terminal hy droxylase (Fig. 1). The activity was not restored by the addition of vitamin C to the incubation mixture and the activity was fully restored by the administration of ascorbic acid 24 hours before sacrifice (28, 29, 40). Thus, vitamin C likely regulates the biosynthesis of the hydroxylat ing enzymes or act as a stabilizer of the enzymes, whereas the demethylating enzymes seems likely to be less sensitively regulated by vitamin C. SUMMARY 1. The effects of vitamin C deficiency on the activities of drug-metabolizing enzymes and electron-transport systems in liver microsomes of guinea-pigs were investigated.

2. creased

The

hydroxylation

by vitamin

hydramine, 3.

of aniline,

C deficiency,

meperidine Moreover,

and

hexobarbital

in contrast,

N-methylaniline

the 0-demethylation

tase, and

The

activity

oxidase

5.

The

6.

These

ficantly 7.

c reductase,

of cytochrome

results

indicate

enzymes

that

not significantly

NADH-ferricyanide

de

diphen

affected. of p-nitroben

affected.

NADPH-neotetrazolium reductase,

reduc

NADPH

oxidase

altered.

P-450

and

the effect

is likely

was markedly

of aminopyrine,

and the reduction

c reductase,

was not significantly

content

drug-metabolizing

were

of NADPH-cytochrome

NADH-cytochrome NADH

zoxazolamine

was not significantly

of p-nitroanisole

zoic acid and p-dimethylaminoazobenzene 4.

and

the N-demethylation

specific

b5 was not significantly of vitamin

and

C deficiency

the hydroxylating

altered. on the microsomal

reaction

is only signi

affected. Thus,

the

mechanism

vitamin

C deficiency

xylating

reaction.

is likely

of the

decrease

involved

in the

in the hydroxylation step

of drugs

of the terminal

oxidase

induced

by

for hydro

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