Toxicology, 8 (1977) 23--32 © Elsevier/North-Holland Scientific Publishers, Ltd.
D I E T A R Y E F F E C T S ON INHIBITION OF RAT HEPATIC MICROSOMAL DRUG-METABOLIZING ENZYMES BY A PESTICIDE (MORESTAN®) *
DANIELLE GAILLARD, GHISLAINE CHAMOISEAU and RO G ER DERACHE Groupe de Recherches sur la Toxic®logic des Aliments et des Boissons, LN.S.E.R.M. - U.87. Universitd Paul Sabatier, 2 Rue Franfois Magendie, 31400 Toulouse (France) (Received November 1st, 1976) (Revision received December 20th, 1976) (Accepted December 31st, 1976)
SUMMARY
Female rats were fed for 21 days on 5 semi-synthetic diets containing 8 or 30% proteins, I or 25% lipids respectively, the control animals being given a diet containing 20% proteins and 5% lipids. The animals on each diet were then subdivided into two subgroups and on the 22nd, 23rd, 24th and 25th days were given an oral dose of 75 mg/kg of Morestan ® in solution in peanut oil (PO) or a dose of oil only. The microsomes were prepared 24 h after the last administration and aniline aromatic hydroxylase, aminopyrine and N-methylaniline N-demethylase activities and cytochrome P-450, protein and R N A levels were measured. Whatever the diet, Morestan ® inhibited N-demethylase activities and decreased the c y t o c h r o m e P-450 level; liver protein and R N A levels and microsomal R N A level increased. The 25% lipid diet alone increased activity of the three enzymes studied, without modifying the cytochrome P-450 level; Morestan ® produced antagonism to this effect in the rats on this diet. The decreased c y t o c h r o m e P-450 level caused by M o r e s t a n V w a s higher in animals on the 8% protein diet.
INTRODUCTION
Low-protein diets are known to decrease enzymatic induction provoked by phenobarbital [1] and 3-methylcholanthrene [2], while induction is * This work was undertaken with financial aid from the Ministry for the Protection of Nature and Environment: research contract No. 73 - 61 (139). Abbreviations: PO, peanut oil.
23
increased in rats o n high-fat diets t h a t are given p h e n o b a r b i t a l [3,4] or 2,3d i m e t h y l q u i n o x a l i n e [5] t r e a t m e n t s at t h e s a m e t i m e . Nevertheless, we f o u n d n o i n f o r m a t i o n c o n c e r n i n g possible i n t e r a c t i o n o f diets a n d substances inhibiting the activities o f m i c r o s o m a l m o n o o x y g e n a s e s . In a p r e v i o u s w o r k [ 6 ] , we s h o w e d t h a t various h e p a t i c m i x e d - f u n c t i o n o x i d a s e s are i n h i b i t e d b y i n t r a p e r i t o n e a l a n d oral a d m i n i s t r a t i o n s o f Morestan ® ( 6 - m e t h y l - 2 , 3 - q u i n o x a l i n e d i t h i o l cyclic-S,S c a r b o n a t e ; o x y t h i o c h i n o x ; q u i n o m e t h i o n a t e ) , w h i c h is an acaricide a n d fungicide n o w w i d e l y u s e d in a g r i c u l t u r e in n u m e r o u s c o u n t r i e s . M o r e s t a n ® also decreases t h e c y t o c h r o m e P - 4 5 0 level at t h e s a m e t i m e . T h e p u r p o s e o f t h e p r e s e n t studies was t o investigate t h e e f f e c t o f Morestan ® in a n i m a l s given i n a d e q u a t e diets. Diets c o n t a i n i n g high- or l o w - p r o t e i n a n d high- o r low-lipid diets w e r e a d m i n i s t e r e d t o rats, c o n t r o l animals receiving a b a l a n c e d diet. T h e possible i n t e r a c t i o n o f the diets a n d t h e orallya d m i n i s t e r e d M o r e s t a n ® on h e p a t i c m i c r o s o m a l d r u g - m e t a b o l i z i n g e n z y m e s y s t e m s was e x a m i n e d . METHODS
Preparation of animals and composition of diets F e m a l e rats ( S p r a g u e - - D a w l e y ) aged a p p r o x . 29 d a y s , average w e i g h t 65 g, w e r e divided i n t o 5 g r o u p s o f 32 animals. A g r o u p was given o n e o f t h e 5 s e m i - s y n t h e t i c diets f o r 21 d a y s ; t h e c o m p o s i t i o n o f t h e diets is s h o w n in T a b l e I. T h e o n l y d i f f e r e n c e b e t w e e n t h e diets is t h e p e r c e n t a g e s o f lipids
TABLE I BASAL DIET COMPOSITIONS WITH ISOCALORICALLY SUBSTITUTED PROTEIN AND LIPID Ingredients
Control a
8% Protein
30% Protein
1% Lipid
25% Lipid
Lactic caseine b Peanut oil c Sucrose Vitamin mixture d Salt mixture d Cellulose
250 50 550 10 100 40
100 50 700 10 100 40
375 50 430 10 100 40
250 10 650 10 100 40
250 250 160 10 100 230
a 20% protein, 5% lipid. b Supplied by Monnet, Paris, France; 80% protein content, defatted acid washed preparation. c Supplied by Institut des Corps Gras, Paris, France; 100% peanut oil, 25.6% linoleic acid content (obtained by gas phase chromatography), without anti-oxidants, stored at --15°C (I peroxide = 0.1 mEq./kg). Safety precaution: to decrease the possibility of peroxidation occurring, the lipid was added to the diet every day and a fresh diet was made available to the experimental animals daily. d Supplied by U.A.R., Villemoisson-sur-Orge, France.
24
(1 or 25%) and proteins (8 or 30%); food and drinking water were given ad libitum and ingesta measured daily. The rats were weighed twice weekly and the weight curve recorded.
Treatment of animals The animals in each group on the same diet were subdivided into two subgroups from the 22nd day onwards. While continuing the same diets, the first subgroup of 16 rats was given 75 mg/kg bodyweight of Morestan ® (Chem Service Inc.) dissolved in pure peanut oil (7.5 mg/ml) by oral administration at 24-h intervals for 4 days; this dose of Morestan ® is equal to 0.025 of the acute oral LDs0 [7]. The rats in the second subgroup served as control subjects for the diet, and were given 10 ml/kg peanut oil under the same experimental conditions. F o o d and drinking water were given ad libitum, the ingesta measured daily and the rats weighed. The rats were decapitated 24 h after the final administration, and the liver removed immediately and weighed; proteins [8] and R N A [9] were measured on an aliquot. Protein standards were carried o u t with fraction V human albumin serum (Nutritional Biochemicals Corp.); RNA was evaluated by sheep liver R N A (Choay).
Evaluation of activities of various hepatic mixed-function oxidases and measurement o f cytochrome P-450, protein and RNA levels in liver microsomes The rat liver was homogenized in 4 vols. (w/v) of i c e , o l d 0.1 M phosphate buffer, pH 7.4, using a motor-driven Potter homogenizer having a closefitting Teflon pestle (about 40 mg of microsomal protein/10 ml of the liver homogenate). Preliminary centrifuging at 9000 g for 15 min at 4°C eliminated mitochondria, nuclei and cellular debris. The supernatant was then centrifuged at 4°C at 105,000 g for 1 h (M.S.E. ultracentrifuge). The pellet containing the microsomes was placed in suspension in ice-cold phosphate buffer at protein concentration of approximately 8 mg/ml. The protein [8] and R N A [9] levels of the microsomes were measured by the same standards as above. Cytochrome P-450 level was also evaluated by using the extinction coefficient 91 c m - ' mM -1 [10]. Microsomal enzyme activity was estimated after incubation in medium containing 0.75 pM NADP, 50 pM glucose-6-phosphate, 0.5 I.U. glucose-6phosphate dehydrogenase, 25 pM C12 Mg, 100 pM nicotinamide, 5 pM NADH and 290/~M potassium phosphate buffer, pH 7.4. Substrate concentrations were 5 pM for aniline, N-methylaniline and aminopyrine. The quantity of microsomal proteins present in the 6 ml incubation medium was approx. 20 mg. Incubation was carried out for 30 min with shaking at 150 oscillations/min in a Gallenkamp apparatus, thermostat at 37°C, air being used as gaseous phase. The metabolites formed from the three substrates used were then m e a s u r e d . Aniiine aromatic hydroxylation was estimated by the appearance of p-aminophenol [ 11 ], N-methylaniline N-demethylation by
25
the formation of formaldehyde [12] and aminopyrine N-demethylation by the formation of 4-aminoantipyrine [ 13]. Results are expressed in nanomoles of metabolite formed during 30-min incubation/mg microsomal proteins.
Expression o f results and statistical calculations The effects of the diet alone, Morestan ® treatment in rats (principal effects) and the combined administration of Morestan ® and diet (interaction) were analyzed for each parameter. It was therefore a 2 × 2 factorial arrangement experiment in which the effects of the diet and Morestan ® administration are compared with control groups each time. Calculations were performed according to the method of orthogonal polynomes [ 14]. RESULTS
Effects o f various diets and oral administration o f Morestan ® on growth, f o o d consumption and rat liver weight Table II shows the mean results of the various parameters of the diets and Morestan ®, and also F values from factorial analysis. The results show that the weight of rats given low-protein diet increased much less than those on the other 4 diets, and that the quantity of food ingested by this group of animals was also less than in other groups throughout the 21 days of the diet. Thus b o t h restriction of food intake and deficiency of protein significantly reduced the b o d y weight gain b u t the rats remained apparently healthy and in good conditions for the 3 weeks of the low-protein diet. Animals on the 5 different diets lost weight during the 4-days Morestan ® treatment; at the same time, they ate less food than control rats. At the end of the treatment, the b o d y weight of the animals treated with Morestan ® was distinctly lower than that of animals given gastric intubation of oil under the same experimental conditions. However, when Morestan ® was administered to animals on the 8% protein diet, the p h e n o m e n o n was potentialized (F = 21.85). Liver weight per 100 g bodyweight increased significantly after Morestan v treatment, whatever the diet. This was also the case of rats fed on 8% protein diet, b u t when Morestan ® was given to them at the s a m e time, there was merely summation of the principal effects and interaction was n o t significant. On the contrary, in the case of the 25% lipid diet, the effect of Morestan® on liver enlargement was increased (F = 6.69). Effect o f oral administration o f Morestan ® on protein and R N A levels in liver and microsomes o f rats on the various diets The results in Table III show that Morestan ® significantly increased protein and R N A levels in the liver, whatever the diet previously given to the
26
b~
-+
-+
--
Control
30% protein
8% p r o t e i n
-+
1% l i p i d
ADMINISTRATIONS
OF
MORESTAN ® ON FOOD
CONSUMPTION,
3 . 6 4 +- 0 . 5 9
4.23 ± 1.06
1.15 + 0.74 a
3.80 ± 0.61
3.59 ± 0.62
15.6 ± 0.4
14.2 ± 0.4
13.1 ± 0.3 a
1 5 . 3 + 0.3
15.3 ± 0.6
+ 1 . 7 0 -+ 1 . 4 5 --4.12 ± 2.83 F = 68.05 b
+ 1.80 + 0.60 --4.16 ± 2.07 F = 79.86 b
--3.12 ± 0.98 F = 41.89 b
+ 0.28 ± 1.51
+ 1.89 ± 0.83 --4.12 ± 0.84 F = 74.80 b
+ 2.16 ÷ 0.72 --4.59 ± 1.59
-+ 0.9 = 14.03 b +- 0.5 = 26.69 b
11.7 ± 0.2 4.7 ± 2.4 F = 19.53 b
1 0 . 3 ± 0.4 3 . 0 ± 1.1 F = 32.08 b
6.3 F 0.8 F
12.8 ± 0.4 5.9 -+ 1.7 F = 25.52 b
13.2 ± 0.2 4 . 3 -+ 2.5
Average food intake g/day/rat
143.4 ± 2.9 1 2 1 . 0 ± 1 .9 F = 127.38 b
1 4 1 . 6 +- 2 .4 119.1 ± 2.0 F = 146.02 b
9 6 . 6 ± 2 .2 F = 502.10 b 8 6 . 7 ± 1.7 F = 92.48 b
1 4 9 . 8 ± 2 .8 1 2 6 . 3 ± 2.1 F = 132.30 b
1 5 0 . 7 ± 2 .4 1 2 2 . 2 ± 1.3
Final body weight g
+- 0 . 1 0 = 23.65 b ± 0.15 = 289.06 b
4.12 ± 0.10 6.14 ± 0.12 F = 431.67 b
3.84 ± 0.05 6.14 ± 0.06 F = 961.07 b
4.78 F 5.72 F
4 . 0 0 +- 0 . 0 4 5 . 9 3 -+ 0 . 1 5 F = 389.89 b
4.19 ± 0.05 6.13 ± 0.09
Liver weight g/100 g body weight
Body weight gain or loss g/day/rat
Body weight gain g/day/rat
Average food intake g/day/rat
A d m i n i s t r a t i o n s o f M o r e s t a n ® or peanut, oil (4 d a y s ) n = 16
Diets (21 days) n = 32
a Significantly different (P < 0.05) from controls by Student's t-test. b S i g n i f i c a n t at 0.01 level.
-+
25% lipid
+
Morestan ®
Dietary groups
PO
of rats studied (n); F values are obtained from factorial analysis (only significant results of
OF DIETARY PROTEIN AND LIPID, AND AND LIVER WEIGHT OF FEMALE RATS
V a l u e s a r e t h e m e a n s + S . E. f o r t h e n u m b e r the principal effects are mentioned).
EFFECTS GROWTH
T A B L E II
b~ 00
III
level.
a Significant
598.59 ± 15.56 922.22 ± 30.74 F = 178.15 a
8 7 5 . 5 9 +- 1 8 . 5 3 F = 255.94 a
+
-+
574.33
-+ 9 . 5 9
± 13.64
--
1% lipid
at,0.01
926.46 ± 33.67 F = 168.65 a
+
25% lipid
601.08
--
8% protein
± 14.90 ± 26.77
597.40 ± 11.25 856.68 ± 31.25 F = 148.70 a
618.08 909.88
Liver protein mg× g liver/100 n=8
-+
@
30% protein
Morestan
-+
groups g rat
of rats studied
Control
Dietary
V a l u e s a r e t h e m e a n s -+ S . E . f o r t h e n u m b e r the principal effects are mentioned).
OF
± 1.81 ± 2.29
± 2.49
± 1.87
82.61 + 1.46 89.40 ± 1.49 F = 16.60 a
89.73 ± 1.47 F = 9.14 a
86.19
96.41 ± 3.28 F = 13.22 a
85.89
83.99 ± 2.75 89.27 ± 2.36 F = 7.97 a
82.40 90.27
Liver RNA mg/g liver protein n=8
(n); F values are obtained
EFFECTS OF DIETARY PROTEIN AND LIPID, AND PO ADMINISTRATIONS AND LIVER MICROSOMAL PROTEIN AND RNA OF FEMALE RATS
TABLE
± 0.32 ± 0.32
± 0.51 ± 0.43
protein
17.69 17.57
16.66
17.69
± 0.30 ± 0.37
± 0.22
÷ 0.35
1 4 . 3 5 -~ 0 . 3 3 F = 52.47 a 16.77 ± 0.29 F = 7.86 a
18.23 17.07
18.56 18.41
PROTEIN
± 4.43 ± 4.34
RNA mg/g protein
161.47 ± 3.46 198.69 ± 4.30 F = 66.49 a
175.72 ÷ 2.79 F = 12.85 a 194.84 ± 2.88 F = 50.68 a
176.75 + 5.09 F = 21.70 a 210.25 ± 4.71 F = 51.89 a
160.33 ± 3.04 191.06 ± 5.12 F = 47.89 a
155.39 188.79
Microsomal microsomal n= 16
RNA,
results of
AND
analysis (only significant
® ON LIVER
factorial
Microsomal mg/g l i v e r n= 16
from
MORESTAN
b0 ¢O
Aniline (Aromatic hydroxylation) n=8
10.53 ± 0.43 13.34 ± 0.81
-+
+
8.27 ± 0.29 6.74 ± 0.14 F = 60.78 a
11.40 + 0.66 F = 10.02 a 8.44 ± 0.34 F = 41.64 a
11.18-+ 0 . 2 4 6.88-+ 0 . 2 1 F = 128.97 a
12.96 ± 1.30 19.17 ± 2.32 F = 4.68 b 18.06 ± 0.69 F = 29.29 a 15.74 ± 0.68
9.63 ± 0.48 7 . 5 5 +- 0 . 1 1 F = 46.69 a
15.74 ± 0.62 16.27 ± 0.74
9.97 ± 0.52 6.91 ± 0.21
Aminopyrine (N-demethylation) n=8
7.15 ± 0.84 5 . 2 7 -+ 0 . 4 4 F = 25.47 a
1 2 . 4 2 -+ 0 . 7 4 F = 21.48 a 4.47 ± 0.35 F = 133.10 a
6.81-+ 1.12 4.01 ± 0.30 F = 25.22 a
8.33 ± 0.66 5.80 ± 0.44 F = 40.19 a
7 . 9 9 -+ 0 . 5 2 4.18 ± 0.29
N-methylaniline (N-demethylation) n=8
Microsomal enzyme activity (nmoles of metabolite formed/mg microsomal protein/30 min)
12.25 ± 1.16 12.50 ± 0.70
ACTIVI-
0 . 5 1 9 -+ 0 . 0 3 1 0.366 ± 0.034 F = 25.97 a
0.431 ± 0.034 F = 17.85 a
0.555 ± 0.038
0.656 ± 0.040 0.370 ± 0.025 F = 56.44 a
0.462 + 0.033 0.441 ± 0.028 F = 7.86 a
0.538 ± 0.024 0.412 ± 0.019
n=16
Cytochrome P-450 (nmole/mg microsomal protein)
of rats studied (n); F values are obtained from factorial analysis (only significant results of
--
a S i g n i f i c a n t at 0.01 level. b S i g n i f i c a n t at 0.05 level.
1% l i p i d
25% lipid
+
-
-+"
30% protein
-
-+
Control
8% p r o t e i n
Morestan ®
Dietary groups
the principal effects are mentioned).
V a l u e s a r e t h e m e a n s + S.E. f o r t h e n u m b e r
EFFECTS OF DIETARY PROTEIN AND LIPID AND PO ADMINISTRATIONS OF MORESTAN ® ON 3 MONOOXYGENASE TIES AND CYTOCHROME P-450 CONTENT OF FEMALE RAT LIVER MICROSOMES
TABLE IV
animals. At the same time, microsomal RNA was also increased by the effect of Morestan ®. The 8% protein diet caused decreased microsomal protein levels compared with the control animals. However, this constituent was increased, as confirmed by interaction {F = 10.04), after Morestan ® treatment in rats given low-protein diet, while it was not modified in animals on the other four diets. Microsomal RNA level increased in animals on 8% protein diet; nevertheless, the main effects of Morestan ® and this diet were only additive. The same p h e n o m e n o n occurred for this parameter with the 25% lipid diet. Evaluations o f activities of some micros®real monooxygenases and cytochrome P-450 level The results given in Table IV show that, whatever the diet, Morestan ® is a powerful inhibitor of aminopyrine and N-methylaniline N-demethylases. At the same time, it very significantly decreases the cytochrome P-450 level in animals on the 5 different diets. Only the 25% lipid diet increased the activity of the three monooxygen ases tested, but it had no effect on cytochrome P-450 level. However, when animals on this diet were treated by Morestan ®, the principal inhibitory effect of Morestan ® was f o u n d in the hepatic microsomal drug-metabolizing enzymes. There was interaction when N-methylaniline was used as Substrate, t h a t is, the inhibitory effect of Morestan v was reinforced in that case (F = 16.50). When Morestan ® was administered at the same time as the 8% protein diet, there was also increased cytochrome P-450 inhibition due to the Morestan ® treatment (F = 8.29). DISCUSSION
Morestan ® administered by oral route to female rats given a balanced diet provokes inhibition of the activity of 2 hepatic mixed-function oxidases, and at the same time a fall in cytochrome P-450 level. The addition of proteins and lipids to the diet does n o t m o d i f y the inhibitory effect of Morestan ® on microsomal monooxygenases, phenomena which are also seen in animals given low protein and lipid diets. Paradoxically, whatever the diet the animals were first given, we observed liver enlargement, increased hepatic protein, RNA and microsomal ttNA after Morestan v treatment. The increased levels of these constituents are difficult to explain, as they generally accompany microsomal enzyme induction [15]. However, experimental examples have been developed in which an increase of the electron microscopicallydemonstrable membranes of the smooth endoplasmic reticulum is accompanied by reduced enzyme activity. For instance, prolonged administration of the pesticide dieldrin to rats has resulted in increased amounts of microsomal proteins and cytochrome P-450 but a reduced activity of some hydroxylating enzymes interpreted as a changed configuration of binding sites [16].
30
Another example is produced by simultaneous administration of phenobarbital w i t h the herbicide 3-amino-l,2,4-triazole, when the increase in membranes is associated with reduced c y t o c h r o m e P-450 concentration [17]. As regards the principal effect of the diets, only the 25% lipid diet activated metabolism of the three substrates used in our experiment. Similar results were f o u n d by Gaillard et al. [18] after rats were given diets containing 20% of various oils for 8 weeks, and by Norred and Wade [19] ; according to the latter authors, the rise in microsomal monooxygenase activities is connected with the increased quantity of corn oil in the diet. However, the inhibitory effect of Morestan ® persists, producing an antagonism. The 1% lipid diet had practically no influence on drug-metabolizing enzyme systems under our experimental conditions. We used a diet containing 1% peanut oil, a level certainly sufficient to maintain the integrity of the microsomal system; diets must be fat-free [4] or contain quantities distinctly lower than 1% [20] to cause decreased microsomai monooxygenase activities. Moreover the rats given 1 ml/100 g of peanut oil did n o t have a 1% fat diet; t h e y had 4 days of 20% calories as fat. According to Kato et al. [21], a diet containing 50% casein has little effect on the oxidase system of microsomes of female rat. As regards the 30% protein diet, we observed no modification due to the effect of the diet itself; however, additional proteins to the diet do n o t prevent the inhibitory effect of Morestan ® on the microsomal enzymatic system. The 8% protein diet had no effect on microsomal enzymes; diets must be protein-free to produce important inhibition of drug-metabolizing enzymes [21,22]; the cytochrome P-450 level is decreased with 3% protein diets [23], and according to Kato et al. [21], a 5% protein diet inhibits some mixed-function oxidases, while a 10% diet does n o t affect them. Nevertheless, the low-protein diet we used produced interaction by increasing the inhibitory effect of Morestan ® on cytochrome P-450. This is n o t to be disregarded in view of the fact that cytochrome P-450 is responsible for activation of molecular oxygen [24] and liaison with the substrate [25,26], thus permitting oxidation of numerous foreign compounds [27]. In summary, Morestan ® inhibits two microsomal N~lemethylases and at the same time decreases the cytochrome P-450 level, whatever the diet used during our experiment. However, when animals are given an 8% protein diet, the decrease in cytochrome P-450 level caused by Morestan ® is greater than in animals on other diets. Morestan ® is known to be an SH-binding agent in vivo and in vitro [28], and it would be possible to explain, at least partly, the inhibitory effect of Morestan ® by this property, since enzymes with SH groups are very a b u n d a n t in the hepatic microsomal system [29]. At the same time, Morestan ® causes loss of bodyweight in the animals. This effect may be due to the action of Morestan ® itself, or the fact that the animals ingested very little food during the 4~lay Morestan ® treatment. Furthermore, food intake restriction can also cause decreased metabolism of foreign compounds [ 30], a restriction that could then reinforce the inhibitory effect of Morestan ®.
31
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
32
W.J. Marshall and A.E.M. Mc Lean, Biochem. Pharmacol., 18 (1969) 153. J.R. Hayes and T.C. Campbell, Biochem. Pharmacol., 23 (1974) 1721. W.J. Marshall and A.E.M. Mc Lean, Biochem. J., 122 {1971) 569. W.P. Norred and A.E. Wade, Biochem. Pharmacol., 22 (1973) 432. M. B~raud, D. Gaillard et R. Derache, J. Eur. Toxicol., 8 (1975) 212. D. Galliard, G. Chamoiseau and R. Derache, Arch. Environ. Contam. Toxicol., in press. T.J. Martin, Agric. Vet. Chem., 4 (1963) 156. J. Paul, in J. Paul (Ed.), Cells and Tissue Culture, Livingstone, Edinburgh, 1961, p. 284. R.W. Wannemacher Jr., W.L. Banks Jr. and W.H. Wunner, Anal. Biochem., .11 (1965) 320. T. Omura and R. Sato, J. Biol. Chem., 239 (1964) 2370. R. Kato and J.R. Gillette, J. Pharmacol. Exp. Ther., 150 (1965) 279. T. Nash, Biochem. J., 55 {1953) 416. B.N. La Du, L. Gaudette, N. Trousof and B.B. Brodie, J. Biol. Chem., 214 (1955) 741. L. Lison, Statistiques Appliqu4es ~ la Biologie Exp4rimentale, Gauthier-Villars, Paris, 1968. A.H. Conney, Pharmacol. Rev., 19 (1967) 317. F. Hutterer, F.M. Klion, A. Wengraf, F. Schaffner and H. Popper, Lab. Invest., 20 (1969) 455. I.H. Raisfeld, P. Bacchin, F. Hutterer and F. Schaffner, Mol. Pharmacol., 6 (1970) 231. D. Galliard, B. Pipy et R. Derache, Ann. Nutr. Aliment., 28 (1974) 17. W.P. Norred and A.E. Wade, Biochem. Pharmacol., 21 (1972) 2887. W.O. Caster, A.E. Wade, F.E. Greene and J.S. Meadows, Life Sci., 9 (1970) 181. R. Kato, T. Oshima and S. Tomizawa, Jpn. J. Pharmaeol., 18 (1968) 356. J.M. Patel and S.S. Pawar, Indian J. Med. Res., 61 (1973) 1492. W.J. Marshall and A.E.M. Me Lean, Biochem. J., 107 (1968) 15 P. T. Omura, R. Sato, D.Y. Cooper, O. Rosenthal and R.W. Estabrook, Fed. Proc. Fed. Am. Soc. Exp. Biol., 24 (1965) 1181. Y. Imai and R. Sato, Biochem. Biophys. Res. Commun., 22 (1966) 620. J.B. Sehenkman, H. Returner and R.W. Estabrook, Mol. Pharmacol., 3 (1967) 113. J.R. Gillette, Adv. Pharmaeol., 4 (1966) 219. G.P. Carlson and K.P. Dubois, J. Pharmacol. Exp. Ther., 173 (1970) 60. K.J. Netter und S. Jenner, Arch. Pharmakol. Exp. Pathol., 255 (1966) 120. M.U.K. Mgbodile and T.C. Campbell, J. Nutr., 102 (19"72) 53.
D I E T A R Y E F F E C T S ON INHIBITION OF RAT HEPATIC MICROSOMAL DRUG-METABOLIZING ENZYMES BY A PESTICIDE (MORESTAN®) *
DANIELLE GAILLARD, GHISLAINE CHAMOISEAU and RO G ER DERACHE Groupe de Recherches sur la Toxic®logic des Aliments et des Boissons, LN.S.E.R.M. - U.87. Universitd Paul Sabatier, 2 Rue Franfois Magendie, 31400 Toulouse (France) (Received November 1st, 1976) (Revision received December 20th, 1976) (Accepted December 31st, 1976)
SUMMARY
Female rats were fed for 21 days on 5 semi-synthetic diets containing 8 or 30% proteins, I or 25% lipids respectively, the control animals being given a diet containing 20% proteins and 5% lipids. The animals on each diet were then subdivided into two subgroups and on the 22nd, 23rd, 24th and 25th days were given an oral dose of 75 mg/kg of Morestan ® in solution in peanut oil (PO) or a dose of oil only. The microsomes were prepared 24 h after the last administration and aniline aromatic hydroxylase, aminopyrine and N-methylaniline N-demethylase activities and cytochrome P-450, protein and R N A levels were measured. Whatever the diet, Morestan ® inhibited N-demethylase activities and decreased the c y t o c h r o m e P-450 level; liver protein and R N A levels and microsomal R N A level increased. The 25% lipid diet alone increased activity of the three enzymes studied, without modifying the cytochrome P-450 level; Morestan ® produced antagonism to this effect in the rats on this diet. The decreased c y t o c h r o m e P-450 level caused by M o r e s t a n V w a s higher in animals on the 8% protein diet.
INTRODUCTION
Low-protein diets are known to decrease enzymatic induction provoked by phenobarbital [1] and 3-methylcholanthrene [2], while induction is * This work was undertaken with financial aid from the Ministry for the Protection of Nature and Environment: research contract No. 73 - 61 (139). Abbreviations: PO, peanut oil.
23
increased in rats o n high-fat diets t h a t are given p h e n o b a r b i t a l [3,4] or 2,3d i m e t h y l q u i n o x a l i n e [5] t r e a t m e n t s at t h e s a m e t i m e . Nevertheless, we f o u n d n o i n f o r m a t i o n c o n c e r n i n g possible i n t e r a c t i o n o f diets a n d substances inhibiting the activities o f m i c r o s o m a l m o n o o x y g e n a s e s . In a p r e v i o u s w o r k [ 6 ] , we s h o w e d t h a t various h e p a t i c m i x e d - f u n c t i o n o x i d a s e s are i n h i b i t e d b y i n t r a p e r i t o n e a l a n d oral a d m i n i s t r a t i o n s o f Morestan ® ( 6 - m e t h y l - 2 , 3 - q u i n o x a l i n e d i t h i o l cyclic-S,S c a r b o n a t e ; o x y t h i o c h i n o x ; q u i n o m e t h i o n a t e ) , w h i c h is an acaricide a n d fungicide n o w w i d e l y u s e d in a g r i c u l t u r e in n u m e r o u s c o u n t r i e s . M o r e s t a n ® also decreases t h e c y t o c h r o m e P - 4 5 0 level at t h e s a m e t i m e . T h e p u r p o s e o f t h e p r e s e n t studies was t o investigate t h e e f f e c t o f Morestan ® in a n i m a l s given i n a d e q u a t e diets. Diets c o n t a i n i n g high- or l o w - p r o t e i n a n d high- o r low-lipid diets w e r e a d m i n i s t e r e d t o rats, c o n t r o l animals receiving a b a l a n c e d diet. T h e possible i n t e r a c t i o n o f the diets a n d t h e orallya d m i n i s t e r e d M o r e s t a n ® on h e p a t i c m i c r o s o m a l d r u g - m e t a b o l i z i n g e n z y m e s y s t e m s was e x a m i n e d . METHODS
Preparation of animals and composition of diets F e m a l e rats ( S p r a g u e - - D a w l e y ) aged a p p r o x . 29 d a y s , average w e i g h t 65 g, w e r e divided i n t o 5 g r o u p s o f 32 animals. A g r o u p was given o n e o f t h e 5 s e m i - s y n t h e t i c diets f o r 21 d a y s ; t h e c o m p o s i t i o n o f t h e diets is s h o w n in T a b l e I. T h e o n l y d i f f e r e n c e b e t w e e n t h e diets is t h e p e r c e n t a g e s o f lipids
TABLE I BASAL DIET COMPOSITIONS WITH ISOCALORICALLY SUBSTITUTED PROTEIN AND LIPID Ingredients
Control a
8% Protein
30% Protein
1% Lipid
25% Lipid
Lactic caseine b Peanut oil c Sucrose Vitamin mixture d Salt mixture d Cellulose
250 50 550 10 100 40
100 50 700 10 100 40
375 50 430 10 100 40
250 10 650 10 100 40
250 250 160 10 100 230
a 20% protein, 5% lipid. b Supplied by Monnet, Paris, France; 80% protein content, defatted acid washed preparation. c Supplied by Institut des Corps Gras, Paris, France; 100% peanut oil, 25.6% linoleic acid content (obtained by gas phase chromatography), without anti-oxidants, stored at --15°C (I peroxide = 0.1 mEq./kg). Safety precaution: to decrease the possibility of peroxidation occurring, the lipid was added to the diet every day and a fresh diet was made available to the experimental animals daily. d Supplied by U.A.R., Villemoisson-sur-Orge, France.
24
(1 or 25%) and proteins (8 or 30%); food and drinking water were given ad libitum and ingesta measured daily. The rats were weighed twice weekly and the weight curve recorded.
Treatment of animals The animals in each group on the same diet were subdivided into two subgroups from the 22nd day onwards. While continuing the same diets, the first subgroup of 16 rats was given 75 mg/kg bodyweight of Morestan ® (Chem Service Inc.) dissolved in pure peanut oil (7.5 mg/ml) by oral administration at 24-h intervals for 4 days; this dose of Morestan ® is equal to 0.025 of the acute oral LDs0 [7]. The rats in the second subgroup served as control subjects for the diet, and were given 10 ml/kg peanut oil under the same experimental conditions. F o o d and drinking water were given ad libitum, the ingesta measured daily and the rats weighed. The rats were decapitated 24 h after the final administration, and the liver removed immediately and weighed; proteins [8] and R N A [9] were measured on an aliquot. Protein standards were carried o u t with fraction V human albumin serum (Nutritional Biochemicals Corp.); RNA was evaluated by sheep liver R N A (Choay).
Evaluation of activities of various hepatic mixed-function oxidases and measurement o f cytochrome P-450, protein and RNA levels in liver microsomes The rat liver was homogenized in 4 vols. (w/v) of i c e , o l d 0.1 M phosphate buffer, pH 7.4, using a motor-driven Potter homogenizer having a closefitting Teflon pestle (about 40 mg of microsomal protein/10 ml of the liver homogenate). Preliminary centrifuging at 9000 g for 15 min at 4°C eliminated mitochondria, nuclei and cellular debris. The supernatant was then centrifuged at 4°C at 105,000 g for 1 h (M.S.E. ultracentrifuge). The pellet containing the microsomes was placed in suspension in ice-cold phosphate buffer at protein concentration of approximately 8 mg/ml. The protein [8] and R N A [9] levels of the microsomes were measured by the same standards as above. Cytochrome P-450 level was also evaluated by using the extinction coefficient 91 c m - ' mM -1 [10]. Microsomal enzyme activity was estimated after incubation in medium containing 0.75 pM NADP, 50 pM glucose-6-phosphate, 0.5 I.U. glucose-6phosphate dehydrogenase, 25 pM C12 Mg, 100 pM nicotinamide, 5 pM NADH and 290/~M potassium phosphate buffer, pH 7.4. Substrate concentrations were 5 pM for aniline, N-methylaniline and aminopyrine. The quantity of microsomal proteins present in the 6 ml incubation medium was approx. 20 mg. Incubation was carried out for 30 min with shaking at 150 oscillations/min in a Gallenkamp apparatus, thermostat at 37°C, air being used as gaseous phase. The metabolites formed from the three substrates used were then m e a s u r e d . Aniiine aromatic hydroxylation was estimated by the appearance of p-aminophenol [ 11 ], N-methylaniline N-demethylation by
25
the formation of formaldehyde [12] and aminopyrine N-demethylation by the formation of 4-aminoantipyrine [ 13]. Results are expressed in nanomoles of metabolite formed during 30-min incubation/mg microsomal proteins.
Expression o f results and statistical calculations The effects of the diet alone, Morestan ® treatment in rats (principal effects) and the combined administration of Morestan ® and diet (interaction) were analyzed for each parameter. It was therefore a 2 × 2 factorial arrangement experiment in which the effects of the diet and Morestan ® administration are compared with control groups each time. Calculations were performed according to the method of orthogonal polynomes [ 14]. RESULTS
Effects o f various diets and oral administration o f Morestan ® on growth, f o o d consumption and rat liver weight Table II shows the mean results of the various parameters of the diets and Morestan ®, and also F values from factorial analysis. The results show that the weight of rats given low-protein diet increased much less than those on the other 4 diets, and that the quantity of food ingested by this group of animals was also less than in other groups throughout the 21 days of the diet. Thus b o t h restriction of food intake and deficiency of protein significantly reduced the b o d y weight gain b u t the rats remained apparently healthy and in good conditions for the 3 weeks of the low-protein diet. Animals on the 5 different diets lost weight during the 4-days Morestan ® treatment; at the same time, they ate less food than control rats. At the end of the treatment, the b o d y weight of the animals treated with Morestan ® was distinctly lower than that of animals given gastric intubation of oil under the same experimental conditions. However, when Morestan ® was administered to animals on the 8% protein diet, the p h e n o m e n o n was potentialized (F = 21.85). Liver weight per 100 g bodyweight increased significantly after Morestan v treatment, whatever the diet. This was also the case of rats fed on 8% protein diet, b u t when Morestan ® was given to them at the s a m e time, there was merely summation of the principal effects and interaction was n o t significant. On the contrary, in the case of the 25% lipid diet, the effect of Morestan® on liver enlargement was increased (F = 6.69). Effect o f oral administration o f Morestan ® on protein and R N A levels in liver and microsomes o f rats on the various diets The results in Table III show that Morestan ® significantly increased protein and R N A levels in the liver, whatever the diet previously given to the
26
b~
-+
-+
--
Control
30% protein
8% p r o t e i n
-+
1% l i p i d
ADMINISTRATIONS
OF
MORESTAN ® ON FOOD
CONSUMPTION,
3 . 6 4 +- 0 . 5 9
4.23 ± 1.06
1.15 + 0.74 a
3.80 ± 0.61
3.59 ± 0.62
15.6 ± 0.4
14.2 ± 0.4
13.1 ± 0.3 a
1 5 . 3 + 0.3
15.3 ± 0.6
+ 1 . 7 0 -+ 1 . 4 5 --4.12 ± 2.83 F = 68.05 b
+ 1.80 + 0.60 --4.16 ± 2.07 F = 79.86 b
--3.12 ± 0.98 F = 41.89 b
+ 0.28 ± 1.51
+ 1.89 ± 0.83 --4.12 ± 0.84 F = 74.80 b
+ 2.16 ÷ 0.72 --4.59 ± 1.59
-+ 0.9 = 14.03 b +- 0.5 = 26.69 b
11.7 ± 0.2 4.7 ± 2.4 F = 19.53 b
1 0 . 3 ± 0.4 3 . 0 ± 1.1 F = 32.08 b
6.3 F 0.8 F
12.8 ± 0.4 5.9 -+ 1.7 F = 25.52 b
13.2 ± 0.2 4 . 3 -+ 2.5
Average food intake g/day/rat
143.4 ± 2.9 1 2 1 . 0 ± 1 .9 F = 127.38 b
1 4 1 . 6 +- 2 .4 119.1 ± 2.0 F = 146.02 b
9 6 . 6 ± 2 .2 F = 502.10 b 8 6 . 7 ± 1.7 F = 92.48 b
1 4 9 . 8 ± 2 .8 1 2 6 . 3 ± 2.1 F = 132.30 b
1 5 0 . 7 ± 2 .4 1 2 2 . 2 ± 1.3
Final body weight g
+- 0 . 1 0 = 23.65 b ± 0.15 = 289.06 b
4.12 ± 0.10 6.14 ± 0.12 F = 431.67 b
3.84 ± 0.05 6.14 ± 0.06 F = 961.07 b
4.78 F 5.72 F
4 . 0 0 +- 0 . 0 4 5 . 9 3 -+ 0 . 1 5 F = 389.89 b
4.19 ± 0.05 6.13 ± 0.09
Liver weight g/100 g body weight
Body weight gain or loss g/day/rat
Body weight gain g/day/rat
Average food intake g/day/rat
A d m i n i s t r a t i o n s o f M o r e s t a n ® or peanut, oil (4 d a y s ) n = 16
Diets (21 days) n = 32
a Significantly different (P < 0.05) from controls by Student's t-test. b S i g n i f i c a n t at 0.01 level.
-+
25% lipid
+
Morestan ®
Dietary groups
PO
of rats studied (n); F values are obtained from factorial analysis (only significant results of
OF DIETARY PROTEIN AND LIPID, AND AND LIVER WEIGHT OF FEMALE RATS
V a l u e s a r e t h e m e a n s + S . E. f o r t h e n u m b e r the principal effects are mentioned).
EFFECTS GROWTH
T A B L E II
b~ 00
III
level.
a Significant
598.59 ± 15.56 922.22 ± 30.74 F = 178.15 a
8 7 5 . 5 9 +- 1 8 . 5 3 F = 255.94 a
+
-+
574.33
-+ 9 . 5 9
± 13.64
--
1% lipid
at,0.01
926.46 ± 33.67 F = 168.65 a
+
25% lipid
601.08
--
8% protein
± 14.90 ± 26.77
597.40 ± 11.25 856.68 ± 31.25 F = 148.70 a
618.08 909.88
Liver protein mg× g liver/100 n=8
-+
@
30% protein
Morestan
-+
groups g rat
of rats studied
Control
Dietary
V a l u e s a r e t h e m e a n s -+ S . E . f o r t h e n u m b e r the principal effects are mentioned).
OF
± 1.81 ± 2.29
± 2.49
± 1.87
82.61 + 1.46 89.40 ± 1.49 F = 16.60 a
89.73 ± 1.47 F = 9.14 a
86.19
96.41 ± 3.28 F = 13.22 a
85.89
83.99 ± 2.75 89.27 ± 2.36 F = 7.97 a
82.40 90.27
Liver RNA mg/g liver protein n=8
(n); F values are obtained
EFFECTS OF DIETARY PROTEIN AND LIPID, AND PO ADMINISTRATIONS AND LIVER MICROSOMAL PROTEIN AND RNA OF FEMALE RATS
TABLE
± 0.32 ± 0.32
± 0.51 ± 0.43
protein
17.69 17.57
16.66
17.69
± 0.30 ± 0.37
± 0.22
÷ 0.35
1 4 . 3 5 -~ 0 . 3 3 F = 52.47 a 16.77 ± 0.29 F = 7.86 a
18.23 17.07
18.56 18.41
PROTEIN
± 4.43 ± 4.34
RNA mg/g protein
161.47 ± 3.46 198.69 ± 4.30 F = 66.49 a
175.72 ÷ 2.79 F = 12.85 a 194.84 ± 2.88 F = 50.68 a
176.75 + 5.09 F = 21.70 a 210.25 ± 4.71 F = 51.89 a
160.33 ± 3.04 191.06 ± 5.12 F = 47.89 a
155.39 188.79
Microsomal microsomal n= 16
RNA,
results of
AND
analysis (only significant
® ON LIVER
factorial
Microsomal mg/g l i v e r n= 16
from
MORESTAN
b0 ¢O
Aniline (Aromatic hydroxylation) n=8
10.53 ± 0.43 13.34 ± 0.81
-+
+
8.27 ± 0.29 6.74 ± 0.14 F = 60.78 a
11.40 + 0.66 F = 10.02 a 8.44 ± 0.34 F = 41.64 a
11.18-+ 0 . 2 4 6.88-+ 0 . 2 1 F = 128.97 a
12.96 ± 1.30 19.17 ± 2.32 F = 4.68 b 18.06 ± 0.69 F = 29.29 a 15.74 ± 0.68
9.63 ± 0.48 7 . 5 5 +- 0 . 1 1 F = 46.69 a
15.74 ± 0.62 16.27 ± 0.74
9.97 ± 0.52 6.91 ± 0.21
Aminopyrine (N-demethylation) n=8
7.15 ± 0.84 5 . 2 7 -+ 0 . 4 4 F = 25.47 a
1 2 . 4 2 -+ 0 . 7 4 F = 21.48 a 4.47 ± 0.35 F = 133.10 a
6.81-+ 1.12 4.01 ± 0.30 F = 25.22 a
8.33 ± 0.66 5.80 ± 0.44 F = 40.19 a
7 . 9 9 -+ 0 . 5 2 4.18 ± 0.29
N-methylaniline (N-demethylation) n=8
Microsomal enzyme activity (nmoles of metabolite formed/mg microsomal protein/30 min)
12.25 ± 1.16 12.50 ± 0.70
ACTIVI-
0 . 5 1 9 -+ 0 . 0 3 1 0.366 ± 0.034 F = 25.97 a
0.431 ± 0.034 F = 17.85 a
0.555 ± 0.038
0.656 ± 0.040 0.370 ± 0.025 F = 56.44 a
0.462 + 0.033 0.441 ± 0.028 F = 7.86 a
0.538 ± 0.024 0.412 ± 0.019
n=16
Cytochrome P-450 (nmole/mg microsomal protein)
of rats studied (n); F values are obtained from factorial analysis (only significant results of
--
a S i g n i f i c a n t at 0.01 level. b S i g n i f i c a n t at 0.05 level.
1% l i p i d
25% lipid
+
-
-+"
30% protein
-
-+
Control
8% p r o t e i n
Morestan ®
Dietary groups
the principal effects are mentioned).
V a l u e s a r e t h e m e a n s + S.E. f o r t h e n u m b e r
EFFECTS OF DIETARY PROTEIN AND LIPID AND PO ADMINISTRATIONS OF MORESTAN ® ON 3 MONOOXYGENASE TIES AND CYTOCHROME P-450 CONTENT OF FEMALE RAT LIVER MICROSOMES
TABLE IV
animals. At the same time, microsomal RNA was also increased by the effect of Morestan ®. The 8% protein diet caused decreased microsomal protein levels compared with the control animals. However, this constituent was increased, as confirmed by interaction {F = 10.04), after Morestan ® treatment in rats given low-protein diet, while it was not modified in animals on the other four diets. Microsomal RNA level increased in animals on 8% protein diet; nevertheless, the main effects of Morestan ® and this diet were only additive. The same p h e n o m e n o n occurred for this parameter with the 25% lipid diet. Evaluations o f activities of some micros®real monooxygenases and cytochrome P-450 level The results given in Table IV show that, whatever the diet, Morestan ® is a powerful inhibitor of aminopyrine and N-methylaniline N-demethylases. At the same time, it very significantly decreases the cytochrome P-450 level in animals on the 5 different diets. Only the 25% lipid diet increased the activity of the three monooxygen ases tested, but it had no effect on cytochrome P-450 level. However, when animals on this diet were treated by Morestan ®, the principal inhibitory effect of Morestan ® was f o u n d in the hepatic microsomal drug-metabolizing enzymes. There was interaction when N-methylaniline was used as Substrate, t h a t is, the inhibitory effect of Morestan v was reinforced in that case (F = 16.50). When Morestan ® was administered at the same time as the 8% protein diet, there was also increased cytochrome P-450 inhibition due to the Morestan ® treatment (F = 8.29). DISCUSSION
Morestan ® administered by oral route to female rats given a balanced diet provokes inhibition of the activity of 2 hepatic mixed-function oxidases, and at the same time a fall in cytochrome P-450 level. The addition of proteins and lipids to the diet does n o t m o d i f y the inhibitory effect of Morestan ® on microsomal monooxygenases, phenomena which are also seen in animals given low protein and lipid diets. Paradoxically, whatever the diet the animals were first given, we observed liver enlargement, increased hepatic protein, RNA and microsomal ttNA after Morestan v treatment. The increased levels of these constituents are difficult to explain, as they generally accompany microsomal enzyme induction [15]. However, experimental examples have been developed in which an increase of the electron microscopicallydemonstrable membranes of the smooth endoplasmic reticulum is accompanied by reduced enzyme activity. For instance, prolonged administration of the pesticide dieldrin to rats has resulted in increased amounts of microsomal proteins and cytochrome P-450 but a reduced activity of some hydroxylating enzymes interpreted as a changed configuration of binding sites [16].
30
Another example is produced by simultaneous administration of phenobarbital w i t h the herbicide 3-amino-l,2,4-triazole, when the increase in membranes is associated with reduced c y t o c h r o m e P-450 concentration [17]. As regards the principal effect of the diets, only the 25% lipid diet activated metabolism of the three substrates used in our experiment. Similar results were f o u n d by Gaillard et al. [18] after rats were given diets containing 20% of various oils for 8 weeks, and by Norred and Wade [19] ; according to the latter authors, the rise in microsomal monooxygenase activities is connected with the increased quantity of corn oil in the diet. However, the inhibitory effect of Morestan ® persists, producing an antagonism. The 1% lipid diet had practically no influence on drug-metabolizing enzyme systems under our experimental conditions. We used a diet containing 1% peanut oil, a level certainly sufficient to maintain the integrity of the microsomal system; diets must be fat-free [4] or contain quantities distinctly lower than 1% [20] to cause decreased microsomai monooxygenase activities. Moreover the rats given 1 ml/100 g of peanut oil did n o t have a 1% fat diet; t h e y had 4 days of 20% calories as fat. According to Kato et al. [21], a diet containing 50% casein has little effect on the oxidase system of microsomes of female rat. As regards the 30% protein diet, we observed no modification due to the effect of the diet itself; however, additional proteins to the diet do n o t prevent the inhibitory effect of Morestan ® on the microsomal enzymatic system. The 8% protein diet had no effect on microsomal enzymes; diets must be protein-free to produce important inhibition of drug-metabolizing enzymes [21,22]; the cytochrome P-450 level is decreased with 3% protein diets [23], and according to Kato et al. [21], a 5% protein diet inhibits some mixed-function oxidases, while a 10% diet does n o t affect them. Nevertheless, the low-protein diet we used produced interaction by increasing the inhibitory effect of Morestan ® on cytochrome P-450. This is n o t to be disregarded in view of the fact that cytochrome P-450 is responsible for activation of molecular oxygen [24] and liaison with the substrate [25,26], thus permitting oxidation of numerous foreign compounds [27]. In summary, Morestan ® inhibits two microsomal N~lemethylases and at the same time decreases the cytochrome P-450 level, whatever the diet used during our experiment. However, when animals are given an 8% protein diet, the decrease in cytochrome P-450 level caused by Morestan ® is greater than in animals on other diets. Morestan ® is known to be an SH-binding agent in vivo and in vitro [28], and it would be possible to explain, at least partly, the inhibitory effect of Morestan ® by this property, since enzymes with SH groups are very a b u n d a n t in the hepatic microsomal system [29]. At the same time, Morestan ® causes loss of bodyweight in the animals. This effect may be due to the action of Morestan ® itself, or the fact that the animals ingested very little food during the 4~lay Morestan ® treatment. Furthermore, food intake restriction can also cause decreased metabolism of foreign compounds [ 30], a restriction that could then reinforce the inhibitory effect of Morestan ®.
31
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
32
W.J. Marshall and A.E.M. Mc Lean, Biochem. Pharmacol., 18 (1969) 153. J.R. Hayes and T.C. Campbell, Biochem. Pharmacol., 23 (1974) 1721. W.J. Marshall and A.E.M. Mc Lean, Biochem. J., 122 {1971) 569. W.P. Norred and A.E. Wade, Biochem. Pharmacol., 22 (1973) 432. M. B~raud, D. Gaillard et R. Derache, J. Eur. Toxicol., 8 (1975) 212. D. Galliard, G. Chamoiseau and R. Derache, Arch. Environ. Contam. Toxicol., in press. T.J. Martin, Agric. Vet. Chem., 4 (1963) 156. J. Paul, in J. Paul (Ed.), Cells and Tissue Culture, Livingstone, Edinburgh, 1961, p. 284. R.W. Wannemacher Jr., W.L. Banks Jr. and W.H. Wunner, Anal. Biochem., .11 (1965) 320. T. Omura and R. Sato, J. Biol. Chem., 239 (1964) 2370. R. Kato and J.R. Gillette, J. Pharmacol. Exp. Ther., 150 (1965) 279. T. Nash, Biochem. J., 55 {1953) 416. B.N. La Du, L. Gaudette, N. Trousof and B.B. Brodie, J. Biol. Chem., 214 (1955) 741. L. Lison, Statistiques Appliqu4es ~ la Biologie Exp4rimentale, Gauthier-Villars, Paris, 1968. A.H. Conney, Pharmacol. Rev., 19 (1967) 317. F. Hutterer, F.M. Klion, A. Wengraf, F. Schaffner and H. Popper, Lab. Invest., 20 (1969) 455. I.H. Raisfeld, P. Bacchin, F. Hutterer and F. Schaffner, Mol. Pharmacol., 6 (1970) 231. D. Galliard, B. Pipy et R. Derache, Ann. Nutr. Aliment., 28 (1974) 17. W.P. Norred and A.E. Wade, Biochem. Pharmacol., 21 (1972) 2887. W.O. Caster, A.E. Wade, F.E. Greene and J.S. Meadows, Life Sci., 9 (1970) 181. R. Kato, T. Oshima and S. Tomizawa, Jpn. J. Pharmaeol., 18 (1968) 356. J.M. Patel and S.S. Pawar, Indian J. Med. Res., 61 (1973) 1492. W.J. Marshall and A.E.M. Me Lean, Biochem. J., 107 (1968) 15 P. T. Omura, R. Sato, D.Y. Cooper, O. Rosenthal and R.W. Estabrook, Fed. Proc. Fed. Am. Soc. Exp. Biol., 24 (1965) 1181. Y. Imai and R. Sato, Biochem. Biophys. Res. Commun., 22 (1966) 620. J.B. Sehenkman, H. Returner and R.W. Estabrook, Mol. Pharmacol., 3 (1967) 113. J.R. Gillette, Adv. Pharmaeol., 4 (1966) 219. G.P. Carlson and K.P. Dubois, J. Pharmacol. Exp. Ther., 173 (1970) 60. K.J. Netter und S. Jenner, Arch. Pharmakol. Exp. Pathol., 255 (1966) 120. M.U.K. Mgbodile and T.C. Campbell, J. Nutr., 102 (19"72) 53.
