Lipid composition and monooxygenase relationship in liver microsomal membranes from rats fed unbalanced diets

Toxicology Letters, 23 (1984) 13-77

13

Elsevier

TOXLett

. 1290

LIPID COMPOSITION LIVER MICROSOMAL DIETS

AND MONOOXYGENASE RELATIONSHIP IN MEMBRANES FROM RATS FED UNBALANCED

(Polychlorinated biphenyls; dietary protein; dietary lipids; drug metabolizing zyme; fluidity; microsomes)

J.F.

NARBONNE,

M.A.

PELISSIER*,

D. BONNAMOUR,

C. BORIN*

en-

and R. ALBRECHT*

Laboratoire de Toxicologic Alimentaire, Avenue des Fact&& 33405 Talence Cedex (France) (Received

March

(Accepted

May 17th,

29th,

1984) 1984)

SUMMARY The cholesterol and phospholipid content of microsomal membrane from rats fed either a high lipid (HI) (30% lard) or a low protein (Lp) diet (6% casein) have been compared with those from rats fed a standard

(St) diet (22% casein,

5% lard).

For each diet, half of the group

was treated

with Phenochlor

DP6. A significant increase in the phospholipid cholesterol ratio was observed in rats fed a high lipid diet or treated with DP6. These effects tend to increase the microsomal membrane fluidity. The protein deficiency

decreased

reticulum

membrane.

the

dent to the enzymic microsomal

phospholipid/cholesterol

The specific form

activity

of cytochrome

ratio

of cytochrome

and

then

the

fluidity

P-450 to hydroxylate

P-450 was closely

correlated

of the endoplasmic

aniline which is indepen-

with the viscosity

status

of the

membrane.

INTRODUCTION

It is well known that changes in dietary components cause an alteration in the rate of metabolism of many xenobiotics by the mammalian liver [l]. These metabolic alterations have been attributed mainly to changes in the level and in the nature of cytochrome P-450 [2]. However Suzuki et al. [3] indicates that the more subtle features of specific drug metabolism enzymes in the membrane of the endoplasmic

*Present addresses: (C.B.) Institut de Physiologie, 2, rue F. Magendie, 31400 Toulouse; R.A.) Laboratoire de Biologie du CNAM, 292, rue St Martin, 75141 Paris (France). Abbreviations:

0378-4274/84/$

AH,

03.00

aniline

hydroxylase;

0 Elsevier

Science

Hl, high lipid; Lp, low protein;

Publishers

B.V.

St, standard.

(M.A.P.

and

74

reticulum might well be affected by the fluidity of the phospholipid bilayer. The main factors that determine the membrane lipids fluidity are the degree of insaturation of phospholipid acyl side chain, the phosphatidyl choline/sphingomyelin ratio and chiefly the phospholipid/cholesterol ratio [4]. It was therefore of interest to determine whether changes in fluidity of the microsomal membrane occur when the phospholipid/cholesterol ratio is modified by changes in dietary components (unbalanced diets and inducers). Moreover, the relationship between monooxygenase activity of cytochrome P-450 and membrane fluidity have been investigated. MATERIALS AND METHODS

Three groups each of 16 Sprague-Dawley rats (89 + 1.1 g) (obtained from Charles River) were respectively fed ad lib. for 6 weeks either a standard (St) diet containing 22% casein and 5% lard, or an Hl diet containing 30% lard, or an Lp diet containing 6% casein. In each group 8 rats were fed a corresponding diet contaminated by 50 ppm of a polychlorinated biphenyl (Phenoclor DP6) within 4 weeks before killing. In comparison with the body weight of the St group (296 + 7 g) the Lp group showed a loss of body weight (- 57%), the Hl group showed a slight increase in body weight (+ 11Vo PC 0.05). In each group, the body weight was not modified by DP6 treatment. All rats were starved overnight before killing and preparation of liver microsomes were carried out as previously described [5]. Microsomal protein was determined by the method of Lowry et al. [6]. Cytochrome P-450 was measured by the method of Omura and Sato [7] and aniline hydroxylase was assayed as described by Imai and Sato [B]. Total lipids and phospholipids were extracted from microsomes by the method of Folch et al. [9]. The phospholipids were separated by thin-layer chromatography on silica gel by the method of Skipski et al. [lo] and the phosphate content was measured by the method of Fiske and Subbarow [ll]. Cholesterol was determined by the method of Siedel et al. [12]. Lipid apparent fluidity of microsomal membrane was determined by measurement of the degree of fluorescence polarization (P) using the fluorophore, 1,6-diphenyl-1,3,5-hexatriene at 37°C [13]. Statistical analysis of the data was carried out by the method of Schwartz [ 141. RESULTS

Cholesterol and phospholipid

content of liver microsomes

Table I shows that in liver microsomes from rats fed the HI diet, no change in phospholipid content occurred despite a significant decrease (- 17%, PC 0.05) in cholesterol content, compared with a control group. Thus the phospholipid/cholesterol molar ratio is increased (+ 21%). In the Lp group there is a significant decrease in both the phospholipid content ( - 40%) and the phospholipid/cholesterol molar

75 TABLE LIPID

I APPARENT

FLUIDITY

EXPERIMENTAL

AND

LIPID

COMPOSITION

OF LIVER

MICROSOMES

FROM

GROUPS

St

Hl

Cholesterol”

62k

Phospholipid Phospholipid

45lk21

448+18

7.27

8.78

4.08

8.65

0.204

0.167

0.248

0.162

cholesterol

molar ratio Index of fluorescence

3

StP

LP

Slf

2

67k3 274k9

69f

HIP 4

596k25

53f

LPP 2

51*

545k23

577+23

10.2

11.3

0.157

2

0.191

polarization anmol/mg

protein.

The values presented

are the mean

+ SD for 8 rats.

ratio (-44%) of liver microsomes. Adding DP6 to the diets increases both the phospholipid content and the phospholipid/cholesterol ratio. The apparent fluidity of microsomal membrane estimated by fluorescence polarization measurement is increased by Hl diet and DP6. Protein deficiency markedly increases the apparent viscosity with or without DP6 (near 20%). Variations of cytochrome P-450 microsomal content and AH activity As shown in Table II the Hl diet does not lead to a significant change of liver cytochrome P-450 concentration but decreases AH activity (- 10% P
II

CYTOCHROME MICROSOMES

P-450 FROM

CONTENT

AND

EXPERIMENTAL St

Cytochrome

P-450

(nmol/mg mic. prot.) Aniline hydroxylase (p/mol/nmol Cyt. P-450/min)

ANILINE

HYDROXYLASE

ACTIVITY

OF

LIVER

GROUPS HI

LP

StP

HlP

LPP

0.927

1.027

0.720

1.684

1.959

k 0.084 946 k58

f 0.062 844 -+64

kO.104 1.152

* 0.061 841

+ 0.079

kO.146

1.450

k64

+48

725 +34

855 +74

DISCUSSION

AND

CONCLUSIONS

The fact that lipid plays an important role in controlling different membrane function is now well established [15]. Increases in the molar ratio of phospholipid to cholesterol are known to increase the apparent microviscosity of lipid bilayers in both model and biological membranes [13]. In our results a significant correlation exists between the values of fluorescence polarization and the phospholipid/cholesterol molar ratio (b = - 57.01, r = 0.779 PcO.05). The changes in microsomal lipid composition in rat fed unbalanced diets have significant influence on the monooxygenase activity. Aniline hydroxylase is independent to the enzymic form of cytochrome P-450 [16] and then it is a good index to investigate the role of membrane lipid parameters on the monooxygenase activities. The results obtained by using unbalanced diets with or without the inducer (DP6) show that an increase in the viscosity of the microsomal membrane (by Lp diet) is related to high AH activity. When rats were fed with diets decreasing the membrane viscosity (Hl or DP6 diets) the ability of cytochrome P-450 to hydroxylate aniline is decreased. Increased membrane fluidity is known to increase the mobility of membrane proteins [17] and may therefore facilitate the interaction between cytochrome P-450 and NADPH-cytochrome P-450 reductase. Then the catalytic activity must be increased by the fluidization of the membrane. Our results show that it is not the case for type II substrates such as aniline. ACKNOWLEDGEMENT

This investigation

received financial support from the DGRST No. 80-G-0902.

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