Gen. Pharmac., 1976, Vol. 7, pp. 415 to 419. Peroamon Press. Printed in Great Britain
DEPRESSION OF D R U G METABOLISM IN LIVER MICROSOMES AFTER TREATING RATS WITH UNSATURATED FATTY ACIDS MATTI LANG Department of Physiology, University of Kuopio, SF-70101, Kuopio 10, Finland (Received 7 June 1976) Abstract--1. Elaidic and linoleic acids were administered to rats intraperitoneally at dose levels of 40 and 200 mg/kg during 5 weeks. Fatty acids had a tendency to decrease both the amount of protein and phosphofipids in liver microsomes. Also the fatty acid composition of the microsomal phospholipids was modified by the fatty acids. 2. Microsomal aryl hydrocarbon hydroxylase and p-nitroanisole-O-demethylase activities were lowered by linoleic acid. The decrease in the enzyme activities was, however, not identical. Also elaidic acid tended to decrease the activity of these enzymes. In addition, a slight decrease in microsomal cytochrome P-450 content and in the activity of NADPH cytochrome c reductase took place. 3. The activity of microsomal UDPglucuronosyltransferase was significantly decreased by both of the fatty acids. 4. The results suggest that unsaturated fatty acids affect the activities of microsomal drug metabolizing enzymes at least partly by modifying the membrane structure. 5. It seems that the effects are substrate specific and to some extent opposite to those of dietary unsaturated triglycerides and cholesterol.
INTRODUCTION
HEPAtiC microsomes contain the enzymes which are involved with both hydroxylation and glucuronidation of foreign compounds. The phospholipid molecules of microsomes are known to be necessary for the catalytic activity of mono-oxygenase complex (Strobel et al., 1970) and for the activity of UDPglucuronosyltransferase, catalyzing the glucuronidation of foreign compounds (Graham & Wood, 1969; Jansen, 1975; H~inninen et al., 1975). If the structure or the protein-lipid interaction of microsomes is somehow altered, e.g. with different types of inducers or with in vitro treatments a change in the enzyme activities takes place (Vainio, 1973; Laitinen et al., 1974). One factor maintaining the microsomal enzyme activities is the composition of dietary lipids. Total fat deficiency of the diet has been found to decrease both the activity of hydroxylation and glucuronidation of foreign compounds in microsomes (Norred & Wade, 1972; Laitinen, 1976). There is evidence that the decrease in enzyme activities is due to changes in the composition of microsomal membrane. On the other hand, we have found that dietary cholesterol elevates the microsomal enzyme activities, especially if it is the only lipid component of the diet, and that the elevation is at least partly due to the modified membrane structure (Laitinen, in press). A few reports concerning the effect of dietary fatty acids on drug metabolism have appeared. It has, however, been difficult to evaluate the effect of a certain fatty acid, because
in most reports the source of fatty acids has been a triglyceride (e.g. lard, corn oil, rapeseed oil and sunflower oil) containing more than one fatty acid (Norred & Wade, 1972; Agradi et al., 1975; Rowe & Wills, 1976; Laitinen et al., 1975a; Hietanen et al., 1975). This study was carried out in order to find out the effects of unsaturated fatty acids on microsomal drug metabolism. Elaidic and linoleic acids were administered as i.p. injections to be sure of the quantity rats received. The effects of unsaturated fatty acids on microsomal drug metabolism have also been compared with the effects of cholesterol which is known to increase the rigidity of biological membranes (Papahadjopoulos et al., 1973).
MATERIALS AND METHODS
Twenty-five male rats weighing 150 +__20g were used. The rats were of the strain Wistar/Af/Han/Mol(Han 67) purchased from Mollegaard Avlslaboratoriet A/S (Denmark) and represent the eighth generation outbred with the rotational mating system in the Laboratory Animal Center of the University of Kuopio. During the experiment the rats were kept in groups of five animals and had free access to tap water and standard rat food (Hankkija Ltd., Finland). Elaidic and linoleic acids were administered to rats intraperitoneaUy as dimethylsulphoxide solutions every second day during 5 weeks. The fatty acids (both from Koch-Light Laboratories, England) were dissolved in dimethylsulphoxide (DMSO) (Merck AG, Darmstadt, Germany) to give solutions of 8 and 40 mg 415
416
MATTI LANG
per ml DMSO. Solutions contained 0.5%° v/v of DL-~-tocopherol (Merck AG, Darmstadt, Germany) to avoid the oxidation of the fatty acid and they were stored under a nitrogen atmosphere. The first group of rats received 40 mg elaidic acid/ks, the second group received 200 mg/kg, third group received 40 mg/linoleic acid/ks and the fourth group 200 mg/linoleic acid/ks. Control group received a respective amount of DMSO containing OL-c~-tocopherol. The rats were killed with a blow on the head and bled by cutting the cervical vessels 24 hr after the last injection. The livers were removed and washed with 0.25 M ice-cold sucrose solution. After weighing, the livers were minced with scissors and washed twice with 10 ml sucrose solution to remove the trapped blood. The homogenization of the livers was carried out by using a Potte~Elvehjem type glass-Teflon homogenizer (five pestle strokes/400 rev per min) to give a 20~o (w/v) suspension in 0.25 M sucrose. The homogenate was centrifuged at 10,000 o for 15min (Sorvall SS1) and the microsomal fraction was harvested from the supernatant by centrifuging at 105,000g for 60 min (MSE 50). Microsomes were suspended into 0.15 M KCI so that 1 ml of suspension corresponded to I g liver and about 25 mg of protein/ml. Trypsin treatment of the microsomes was carried out as described earlier by H~inninen & Puukka (1970), using trypsin, type III, from Sigma Chemical Co., St Louis, U.S.A., and stopping the reaction with trypsin inhibitor (type II-O, Sigma). The microsomal protein content was determined using biuret method (Gornall et al., 1949), bovine serum albumin (Sigma) as standard protein. After measuring, the samples were made colourless with the aid of KCN and the background was determined by measuring once more at 555 nm. Microsomal phospholipid content was measured with the method of Bartlett (1959), modified by Laitinen et al. (1974). The amount of phospholipids was calculated as mg lecithin (Sigma type II-E, mol. wt 734) per g liver. The cholesterol content of microsomes was measured as described by Abell et al. (1952) and modified by Anderson & Keys (1956). Fatty acid composition of microsomal phospholipids was analyzed as described by Laitinen (1976a). Microsomal NADPH cytochrome c reductase (E.C. 1.6.2.4) activity was determined by measuring the reduction of cytochrome c from the horse heart (Sigma, type III) at 550nm in a Beckman 24 spectrophotometer
(Phillips & Langdon, 1962). Aryl hydrocarbon hydroxylase (E.C. 1.14.14.2) activity was measured as described earlier by Wattenberg et al. (1962) and modified by Nebert & Gelboin (1968) and Laitinen et al. (1975b), using a Perkin Elmer MPF 3 A spectrofotofluorometer to measure the amount of hydroxylated benzpyrene. p-Nitroanisole-O-demethylase activity was measured according to Netter (1960) by measuring the formation of p-nitrophenol at 420 nm with a Beckman 24 spectrophotometer. Microsomal cytochrome P-450 content was measured as described earlier by Omura & Sato (1964). Microsomes were suspended to 0.1 M phosphate buffer, pH 7.4, to give about 2 mg protein/ml. UDPglucuronosyltransferase activity in microsomes was determined using p-nitrophenol as aglycone (Isselbacher, 1956; H~inninen, 1968). Student's t-test was used to calculate the statistical significance of the results. The P-values less than 0.05 were considered significant.
RESULTS There were no statistically significant differences in the weight gain of the rats receiving fatty acids when compared to the control rats. N o r was there any difference in the liver per body weight ratio between the two groups. Elaidic and linoleic acids did not cause any significant change in the amount of microsomal protein, phospholipid and cholesterol (Table 1); however, both elaidic and linoleic acid had a tendency to decrease the amounts of microsomal protein and phospholipids. This decrease can be seen both in native and trypsin-treated microsomes (Table 1). F r o m Table 2 it can be seen that the pretreatment of rats with elaidic or linoleic acids did not cause any profound changes in fatty acid composition of microsomal phospholipids. Some minor changes, however, took place. The amount of palm±tic acid has increased when the rats received linoleic acid 40mg/kg. A decrease in the amount of palmitoleic acid occurred, when rats were treated with elaidic
Table 1. The protein, phospholipid and cholesterol contents of hepatic microsomes after treating rats with elaidic and linoleic acids. Values are expressed as means +, standard error of the means and they are compared with the control group by using Student's t-test. Each group contained five animals Protein mg/tissue wet wt
Control Elaidic (40ms/ks) Elaidic (200mg/kg) Linoleic (40ms/ks) Linoleic (200 mg/kg)
Phospholipid m g / g tissue wet wt Cholesterol mg/g tissue wet wt
Native microsomes
Trypsintreated microsomes
Native microsomes
Trypsintreated microsomes
Native microsomes
Trypsintreated microsomes
26.1 +, 1.65
13.1 +, 0.59
6.37 +, 0.30
4.20 +, 0.16
0.29 ± 0.01
0.20 +, 0.01
24.7 +, 1.54
11.8 + 0.39
6.15 +, 0.43
4.37 +, 0.69
0.27 +, 0.01
0.18 +, 0.02
25.2 +, 1.52
12.3 +, 0.56
6.04 +, 0.21
4.04 +, 0.14
0.31 +, 0.02
0.22 ± 0.0l
26.0 _ 1.35
12.9 +, 0.38
6.29 +, 0.34
4.04 +, 0.39
0.29 +, 0.02
0.20 ± 0.01
24.2 +, 1.58
11.8 +, 0.77
5.59 +, 0.30
4.06 +__0.43
0.31 +, 0.01
0.22 +, 0.01
Unsaturated fatty acids and hepatic drug metabolism
417
Table 2. The mean values of the fatty acid composition in the rat liver microsomes expressed as percentage of the total amount bound to phospholipids. Rats were treated with elaidic or linoleic acids
Control Elaidic (40 mg/kg) Elaidic (200 mg/kg) Linoleic (40 mg/kg) Linoleic (200 mg/kg)
Palm±tic (16:0)
Palmitoleic (16:1)
Stearic (18:0)
Oleic (18:1)
Linoleic (18:2)
Tetraeicosaenoic (20:4)
20.4
4.6
31.5
9.4
15.4
17.9
20.4
1.2
28.6
8.7
18.5
22.7
20.8
3.4
30.9
10.7
16.6
16.9
25.1
5.2
29.7
9.8
15.1
14.9
20.4
0.5
29.6
9.3
18.4
20.8
acid (40mg/kg) or linoleic acid (200mg/kg). The amount of stearic acid decreased slightly when rats were treated with elaidic or linoleic acids. Elaidic acid (40mg/kg) and linoleic acid (200 mg/kg) elevated the amount of microsomal linoleic acid. The amount of tetraeicosaenoic acid (arachidonic acid) increased, when rats were given 40 mg/kg elaidic acid or 200 mg/kg linoleic acid. A decrease in this amount could, however, be detected after giving the rats 40 mg/kg linoleic acid. The activities of microsomal N A D P H cytochrome c reductase, aryl hydrocarbon hydroxylase, p-nitroanisole-O-demethylase and the content of cytochrome P-450 tended to be reduced due to elaidic and linoleic acid treatments of rats (Table 3). The reduction was, however, statistically significant only in the case of aryl hydrocarbon hydroxylase, when rats received linoleic acid 40 mg/kg, and in the case of p-nitroanisole-O-demethylase (specific activity) when rats received li~aoleic acid 200 mg/kg (Table 3). In native microsomes the activity of microsomal
UDPglucuronosyltransferase was affected by elaidic and linoleic acids. Both doses of elaidic acid caused a highly significant reduction in the measurable enzyme activity when calculated on fresh liver basis (Table 4). Linoleic acid also caused a significant reduction in the activity of UDPglucuronosyltransferase, when calculated on fresh liver basis. The effect was, however, slightly weaker than that of elaidic acid (Table 4). The specific activity of UDPglucuronosyltransferase was found to be reduced to some extent in native microsomes when rats received elaidic or linoleic acids. This reduction was, however, significant only in the case of elaidic acid at the dose level of 200 mg/kg (Table 4). As expected from earlier results, trypsin treatment strongly elevated the activity of microsomal UDPglucuronosyltransferase (Table 4). It can also be seen from Table 4 that in trypsin-treated microsomes the activity of UDPglucuronosyltransferase was clearly lowered after treating the rats with elaidic or linoleic acids, In trypsin-treated microsomes unlike in native microsomes, a significant reduction
Table 3. The activities of NADPH cytochrome c reductase (as #moles cytochrome c reduced/min), aryl hydrocarbon hydroxylase (as nmoles hydroxylated benzpyrene formed/min), and p-nitroanisole-O-demethylase (as nmoles p-nitrophenol formed/min) and the cytochrome P-450 content (as nmoles) in rat liver microsomes after treating the rats with elaidic or linoleic acids. Column A represents the activity/g tissue wet weight and column B represents the specific activity. The means and standard errors of the means are expressed. Statistical analysis of the results have been performed with regard to the control group by using Student's t-test. Each group contained five animals NADPH cytochrome c reductase
Control Elaidic (40 mg/kg) Elaidic (200 mg/kg) Linoleic (40 mg/kg) Linoleic (200 mg/kg)
Aryl hydrocarbon hydroxylase
p-nitroanisole-Odemethylase
Cytochrome P-450
A
B
A
B
A
B
A
B
10.3 + 1.6 9.4 ±_ 1.8 10.1 ± 1.1 7.6 + 0.9 9.5 ± 1.8
0.40 + 0.06 0.39 + 0.08 0.41 ± 0.05 0.30 ± 0.04 0.39 ± 0.07
0.69 _+ 0.11 0.60 ± 0.10 0.64 ± 0.10 0.26t ± 0.06 0.56 ± 0.14
0.027 +__0.005 0.024 +_ 0.004 0.025 + 0.004 0.010" + 0.003 0.023 ± 0.005
9.2 + 1.3 8.0 ___0.2 7.0 ± 0.3 7.9 ± 0.4 6.4 ± 0.6
0.40 + 0.05 0.31 ± 0.03 0.28 ± 0.02 0.30 ± 0.01 0.27* ± 0.02
13.2 + 1.1 11.8 ± 1.3 11,3 + 0.6 10,7 ± 1.5 10.9 ± 1.4
0.51 + 0.04 0.48 ± 0.05 0.45 ± 0,02 0.42 ± 0,07 0.45 ± 0.05
* P < 0.05, t P < 0.01.
418
MA'rrl LANG Table 4. The activity of UDPglucuronosyltransferase, as nmoles p-nitrophenol conjugated/min, in rat liver microsomes after treating the rats with elaidic or linoleic acids. Column A represents the activity/g tissue weight and column B represents the specific activity. The means and standard error of the means are expressed: statistical analysis of the results has been performed with regard to the control group by using Student's t-test. Each group contained five animals Native microsomes
Control Elaidic (40 mg/kg) Elaidic (200 mg/kg) Linoleic (40 mg/kg) Linoleic (200 mg/kg)
Trypsin-treated microsomes
A
B
A
B
36.0 ± 1.0
1.35 ± 0.10
161.7 ± 6.9
12.0 + 0.5
28.2 ± 0.8~
1.16 ± 0.07
120.0 ± 6.1t
9.6 ± 0.9*
26.7 ± 1.2~
0.99 ± 0.08*
104.9 ± 4.3~
8.5 ± 0.6?
31.3 ± 1.2"
1.21 ± 0.03
120.6 ± 7.0f
9.4 ± 0.6f
28.3 + 2.0t
1.19 ± 0.11
112.0 ± 10.7~"
10.6 ± 0.3*
* P < 0.05, f P < 0.01, ~ P < 0.001. in the specific activity of UDPglucuronosyltransferase could be measured in every group receiving fatty acids (Table 4). DISCUSSION According to the present results, it seems that unsaturated fatty acids are able to lower the capacity of microsomal drug metabolizing enzymes, both in the mono-oxygenase system and in glucuronidation. In addition to this overall tendency, it can be seen that changes in the enzyme activities caused by fatty acids are not identical. Accordingly, the activity of aryl hydrocarbon hydroxylase is decreased most when linoleic acid (40 mg/kg) is given to rats, p-nitroanisole-Odemethylase activity decreased most when rats received linoleic acid (200 mg/kg) and the greatest decrease in UDPglucuronosyltransferase activity was detected after giving rats elaidic acid (200 mg/kg). Norred & Wade (1972) have found that the catalytic activity of rat liver microsomal mono-oxygenase system increases if a fat-free diet is supplemented with corn oil. In addition, Rowe & Wills (1976) have found that an increase in the unsaturation degree of dietary triglycerides leads to an elevation in the activity of microsomal oxidative demethylation. Similarly, Agradi et al. (1975) have shown that the hydroxylat±on rate of both benzpyrene and aniline is elevated in rat liver microsomes if the average unsaturation degree of the dietary triglycerides is increased. From the reports mentioned above it is, however, difficult to evaluate whether the effect of dietary triglycerides on drug metabolism is due to a certain fatty acid or due to the combined effect of all the fatty acids plus glycerol. When the present results are compared to earlier reports it seems, however, that unsaturated fatty acids act quite differently on microsomal drug metabolism than dietary unsaturated triglycerides. The effect of arachidonic acid (as ethyl arachidonate) and docosa-
hexaenoic acid (as menhaden oil) on microsomal drug metabolism has been studied by Wade et al. (1972). Various doses of acids were given per orally. It was found that both aniline hydroxylase and hexobarbital oxidase activities in microsomes were decreased to various extent depending on the dose (reference group received corn oil instead of fatty acids). This is in agreement with our results, indicating that unsaturated fatty acids are able to decrease the activity of microsomal hydroxylation reactions at least towards some substrates. The question of the possible mechanism by which fatty acids act on the activities of microsomal drug metabolizing enzymes is difficult to answer. According to the results presented here, fatty acids were able to decrease slightly the amount of microsomal phospholipids. It can also be seen that changes in the activities of microsomal p-nitroanisole-O-demethylase and UDPglucuronosyltransferase but not aryl hydrocarbon hydroxylase correlate well with the changes in the amount of microsomal phospholipids. Although the changes in the amount of microsomal phospholipids were not statistically significant they might, however, be enough to cause significant changes in the activities of some drug metabolizing enzymes. On the other hand, it can be seen that elaidic and linoleic acids were able to modify to some extent the fatty acid composition of phospholipid in microsomes. This might also be one factor in regulating the microsomal enzyme activities towards some substrates (for example, benzpyrene). It is interesting to notice that after trypsin treatment, which is presumed to peel off proteins from the outer layers of microsomal membranes, and reveal UDPglucuronosyltransferase (H~inninen & Puukka, 1970), the decrease in the specific activity of UDPglucuronosyltransferase is more evident when compared to the decrease in native microsomes. It should also be noticed that in trypsin-treated microsomes, changes in UDPglucuronosyltransferase activity do not correlate well
Unsaturated fatty acids and hepatic drug metabolism with the changes in the amount of microsomal phospholipids (unlike in the case of native microsomes). This could mean that the catalytic activity of U D P glucuronosyltransferase is highly dependent on the quality of its phospholipid environment. In our earlier studies we have found that dietary cholesterol is able to increase the activities of microsomal drug metabolizing enzymes. The fatty acid composition of microsomal phospholipids is also altered due to dietary cholesterol (Laitinen, in press) and the changes are partly opposite to those caused by elaidic or linoleic acids. Thus dietary cholesterol tended to decrease the amount of microsomal arachidonic and linoleic acids and increase the amount of palmitoleic acid while the dose of 200 mg/kg linoleic acid and 40 mg/kg elaidic acid had an opposite effect on the amount of these fatty acids in microsomes. The results suggest that unsaturated fatty acids may decrease the activity of microsomal drug metabolizing enzymes, partly by lowering the amount of microsoreal phospholipids and partly by modifying the fatty acid composition of microsomal phospholipids. The effects are to some extent opposite to those caused by dietary cholesterol.
REFERENCES
ABELL L. L., LEVY B. B., BRODIE B. B. & KENDALL F. E. (1952) A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. biol. Chem. 195, 357-366. AGRADI E., SPAGNUOLOC. & CALLI C. (1975) Dietary lipids and aniline and benzpyrene hydroxylations in liver microsomes. Pharmac. Res. Commun. 7, 469-480. ANDERSON J. T. & KEYS A. (1956) Cholesterol in serum and lipoprotein fraction. Clin. Chem. 2, 145-159. BARTLETTG. R. (1959) Phosphorous assay in column chromatography. J. biol. Chem. 234, 466-468. GORNALL A. G., BARDAWILLC. J. & DAVID M. M. (1949) Determination of serum proteins by means of the biuret reaction. J. biol. Chem. 177, 751-766. GRAHAM A. B. & WOOD G. C. (1969) The phospholipid dependence of UDPglucuronosyltransferase. Biochem. biophys. Res. Commun. 37, 567-575. HIETANEN E., LAITINEN M., VAINIO H. t~ HANNINEN O. (1975) Dietary fats and properties of endoplasmic reticulum II. Lipids 10, 467-472. HT~NNINENO. (1968) On the metabolic regulation in the glucuronic acid pathway in the rat tissues. Ann. acad, sci. fenn. A2 (142), 1-96. H~NNINEN O., LANGM. & KOIVUSAARIU. (1975) UDPglucuronosyltransferase: partial separation from other membrane components and reactivation. IUPHAR Satellite Symposium on Active Intermediates: Formation, Toxicity and Inactivation, Turku, Finland. HA.NNINENO. • PUUKKA R. (1970) Effect of digitonin on UDPglucuronosyltransferase in microsomal membranes. Finnish chem. J. 43, 451-456. ISSELBACHERK. J. (1956) Enzymatic mechanism of hormone metabolism. II. Mechanism of hormonal glucur-
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onide formation. Rec. Progr. Hormonal Res. Commun. 12, 134-145. JANSEN P. (1975) Studies on UDPglucuronosyltransferase, the formation of bilirubin mono- and diglucuronide and p-nitrophenyl glucuronide. Academic dissertation, Nijmegen. LAITINEN M. (1976) Hepatic and duodenal drug metabolism in the rat during fat deficiency. Gen. Pharmac. 7, in press. LAITINENM. (in press) Enhancement of hepatic drug metabolism with dietary cholesterol in rat. Acta pharmac, tox. 38. LAITINEN M., HIETANEN E., VAIN10 H. & H~-NNINEN O. (1975a) Dietary fats and properties of endoplasmic reticulum: 1. Lipids 10, 461-466. LAmNEN M., LANG M., HIETANENE. & VAINIOH. (1975b) Enhancement of hepatic drug biotransformation rate by polychlorinated biphenyls in rats fed cholesterol-rich diet. Toxicology 5, 79-88. LAmNEN M., LANG M. & H~NNINEN O. (1974) Changes in the protein-lipid interaction in rat liver microsomes after pretreatment of the rat with barbiturates and polycyclic hydrocarbons. Int. J. Biochem. 5, 747-751. NE~ERTD. W. & GELBOINH. V. (1968) Substrate inducible microsomal aryl hydrocarbon hydroxylase. II. Cellular reponses during enzyme induction. J. biol. Chem. 243, 6242-6249. NETTER K. J. (1960) Line Methode zur direkten Messung der O-Demethylierung in Lebermikrosomen und ihre Andwendung auf die Mikrosom~nhemm-wirkung yon SKF 525-A. Naunyn-Schmiedebero's Arch. Pharmac. 238, 292-300. NORRED W. P. & WADE A. E. (1972) Dietary fatty acid induced alterations of hepatic microsomal drug metabolism. Biochem. Pharmac. 21, 2887-2897. OMURAT. & SATOR. (1964) The carbon monoxide binding pigment of liver microsomes. J. biol. Chem. 239, 2379-2385. PAPAHADJOPOULOS D., COV~DEN M. & KIMELBERG H. (1973) Role of cholesterol in membranes. Effects on phospholipid-protein interactions membrane permeability and enzyme activity. PHILLIPS A. H. t~ LANGDON R. G. (1962) Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization and kinetic studies. J. biol. Chem. 237, 2652-2660. ROWE L. & WILLS E. D. (1976) The effect of dietary lipids and vitamin E on lipid peroxide formation, cytochrome P-450 and oxidative demethylation in the endoplasmic reticulum. Biochem. Pharmac. 25, 175-179. STROBEL H. W., LU A. Y. H., HEIDEMA J. & COON M. J. (1970) Phosphatidylcholine requirement in the enzymic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation. J. biol. Chem. 245, 4851-4854. VAINIO H. (1973) On the topology and synthesis of drug metabolizing enzymes in hepatic endoplasmic reticulum. Academic dissertation, Turku. WADE A. E., Wu B. & CASTERW. O. (1972) Relationship of dietary essential fatty acid consumption to hepatic drug, hydroxylation. Pharmacology 7, 305-314. WATTE~EV,G L. W., LEONG J. L. & STRAND P. J. (1962) Benzpyrene hydroxylase activity in the gastrointestinal tract. Cancer Res. 22, 1120-1125.
DEPRESSION OF D R U G METABOLISM IN LIVER MICROSOMES AFTER TREATING RATS WITH UNSATURATED FATTY ACIDS MATTI LANG Department of Physiology, University of Kuopio, SF-70101, Kuopio 10, Finland (Received 7 June 1976) Abstract--1. Elaidic and linoleic acids were administered to rats intraperitoneally at dose levels of 40 and 200 mg/kg during 5 weeks. Fatty acids had a tendency to decrease both the amount of protein and phosphofipids in liver microsomes. Also the fatty acid composition of the microsomal phospholipids was modified by the fatty acids. 2. Microsomal aryl hydrocarbon hydroxylase and p-nitroanisole-O-demethylase activities were lowered by linoleic acid. The decrease in the enzyme activities was, however, not identical. Also elaidic acid tended to decrease the activity of these enzymes. In addition, a slight decrease in microsomal cytochrome P-450 content and in the activity of NADPH cytochrome c reductase took place. 3. The activity of microsomal UDPglucuronosyltransferase was significantly decreased by both of the fatty acids. 4. The results suggest that unsaturated fatty acids affect the activities of microsomal drug metabolizing enzymes at least partly by modifying the membrane structure. 5. It seems that the effects are substrate specific and to some extent opposite to those of dietary unsaturated triglycerides and cholesterol.
INTRODUCTION
HEPAtiC microsomes contain the enzymes which are involved with both hydroxylation and glucuronidation of foreign compounds. The phospholipid molecules of microsomes are known to be necessary for the catalytic activity of mono-oxygenase complex (Strobel et al., 1970) and for the activity of UDPglucuronosyltransferase, catalyzing the glucuronidation of foreign compounds (Graham & Wood, 1969; Jansen, 1975; H~inninen et al., 1975). If the structure or the protein-lipid interaction of microsomes is somehow altered, e.g. with different types of inducers or with in vitro treatments a change in the enzyme activities takes place (Vainio, 1973; Laitinen et al., 1974). One factor maintaining the microsomal enzyme activities is the composition of dietary lipids. Total fat deficiency of the diet has been found to decrease both the activity of hydroxylation and glucuronidation of foreign compounds in microsomes (Norred & Wade, 1972; Laitinen, 1976). There is evidence that the decrease in enzyme activities is due to changes in the composition of microsomal membrane. On the other hand, we have found that dietary cholesterol elevates the microsomal enzyme activities, especially if it is the only lipid component of the diet, and that the elevation is at least partly due to the modified membrane structure (Laitinen, in press). A few reports concerning the effect of dietary fatty acids on drug metabolism have appeared. It has, however, been difficult to evaluate the effect of a certain fatty acid, because
in most reports the source of fatty acids has been a triglyceride (e.g. lard, corn oil, rapeseed oil and sunflower oil) containing more than one fatty acid (Norred & Wade, 1972; Agradi et al., 1975; Rowe & Wills, 1976; Laitinen et al., 1975a; Hietanen et al., 1975). This study was carried out in order to find out the effects of unsaturated fatty acids on microsomal drug metabolism. Elaidic and linoleic acids were administered as i.p. injections to be sure of the quantity rats received. The effects of unsaturated fatty acids on microsomal drug metabolism have also been compared with the effects of cholesterol which is known to increase the rigidity of biological membranes (Papahadjopoulos et al., 1973).
MATERIALS AND METHODS
Twenty-five male rats weighing 150 +__20g were used. The rats were of the strain Wistar/Af/Han/Mol(Han 67) purchased from Mollegaard Avlslaboratoriet A/S (Denmark) and represent the eighth generation outbred with the rotational mating system in the Laboratory Animal Center of the University of Kuopio. During the experiment the rats were kept in groups of five animals and had free access to tap water and standard rat food (Hankkija Ltd., Finland). Elaidic and linoleic acids were administered to rats intraperitoneaUy as dimethylsulphoxide solutions every second day during 5 weeks. The fatty acids (both from Koch-Light Laboratories, England) were dissolved in dimethylsulphoxide (DMSO) (Merck AG, Darmstadt, Germany) to give solutions of 8 and 40 mg 415
416
MATTI LANG
per ml DMSO. Solutions contained 0.5%° v/v of DL-~-tocopherol (Merck AG, Darmstadt, Germany) to avoid the oxidation of the fatty acid and they were stored under a nitrogen atmosphere. The first group of rats received 40 mg elaidic acid/ks, the second group received 200 mg/kg, third group received 40 mg/linoleic acid/ks and the fourth group 200 mg/linoleic acid/ks. Control group received a respective amount of DMSO containing OL-c~-tocopherol. The rats were killed with a blow on the head and bled by cutting the cervical vessels 24 hr after the last injection. The livers were removed and washed with 0.25 M ice-cold sucrose solution. After weighing, the livers were minced with scissors and washed twice with 10 ml sucrose solution to remove the trapped blood. The homogenization of the livers was carried out by using a Potte~Elvehjem type glass-Teflon homogenizer (five pestle strokes/400 rev per min) to give a 20~o (w/v) suspension in 0.25 M sucrose. The homogenate was centrifuged at 10,000 o for 15min (Sorvall SS1) and the microsomal fraction was harvested from the supernatant by centrifuging at 105,000g for 60 min (MSE 50). Microsomes were suspended into 0.15 M KCI so that 1 ml of suspension corresponded to I g liver and about 25 mg of protein/ml. Trypsin treatment of the microsomes was carried out as described earlier by H~inninen & Puukka (1970), using trypsin, type III, from Sigma Chemical Co., St Louis, U.S.A., and stopping the reaction with trypsin inhibitor (type II-O, Sigma). The microsomal protein content was determined using biuret method (Gornall et al., 1949), bovine serum albumin (Sigma) as standard protein. After measuring, the samples were made colourless with the aid of KCN and the background was determined by measuring once more at 555 nm. Microsomal phospholipid content was measured with the method of Bartlett (1959), modified by Laitinen et al. (1974). The amount of phospholipids was calculated as mg lecithin (Sigma type II-E, mol. wt 734) per g liver. The cholesterol content of microsomes was measured as described by Abell et al. (1952) and modified by Anderson & Keys (1956). Fatty acid composition of microsomal phospholipids was analyzed as described by Laitinen (1976a). Microsomal NADPH cytochrome c reductase (E.C. 1.6.2.4) activity was determined by measuring the reduction of cytochrome c from the horse heart (Sigma, type III) at 550nm in a Beckman 24 spectrophotometer
(Phillips & Langdon, 1962). Aryl hydrocarbon hydroxylase (E.C. 1.14.14.2) activity was measured as described earlier by Wattenberg et al. (1962) and modified by Nebert & Gelboin (1968) and Laitinen et al. (1975b), using a Perkin Elmer MPF 3 A spectrofotofluorometer to measure the amount of hydroxylated benzpyrene. p-Nitroanisole-O-demethylase activity was measured according to Netter (1960) by measuring the formation of p-nitrophenol at 420 nm with a Beckman 24 spectrophotometer. Microsomal cytochrome P-450 content was measured as described earlier by Omura & Sato (1964). Microsomes were suspended to 0.1 M phosphate buffer, pH 7.4, to give about 2 mg protein/ml. UDPglucuronosyltransferase activity in microsomes was determined using p-nitrophenol as aglycone (Isselbacher, 1956; H~inninen, 1968). Student's t-test was used to calculate the statistical significance of the results. The P-values less than 0.05 were considered significant.
RESULTS There were no statistically significant differences in the weight gain of the rats receiving fatty acids when compared to the control rats. N o r was there any difference in the liver per body weight ratio between the two groups. Elaidic and linoleic acids did not cause any significant change in the amount of microsomal protein, phospholipid and cholesterol (Table 1); however, both elaidic and linoleic acid had a tendency to decrease the amounts of microsomal protein and phospholipids. This decrease can be seen both in native and trypsin-treated microsomes (Table 1). F r o m Table 2 it can be seen that the pretreatment of rats with elaidic or linoleic acids did not cause any profound changes in fatty acid composition of microsomal phospholipids. Some minor changes, however, took place. The amount of palm±tic acid has increased when the rats received linoleic acid 40mg/kg. A decrease in the amount of palmitoleic acid occurred, when rats were treated with elaidic
Table 1. The protein, phospholipid and cholesterol contents of hepatic microsomes after treating rats with elaidic and linoleic acids. Values are expressed as means +, standard error of the means and they are compared with the control group by using Student's t-test. Each group contained five animals Protein mg/tissue wet wt
Control Elaidic (40ms/ks) Elaidic (200mg/kg) Linoleic (40ms/ks) Linoleic (200 mg/kg)
Phospholipid m g / g tissue wet wt Cholesterol mg/g tissue wet wt
Native microsomes
Trypsintreated microsomes
Native microsomes
Trypsintreated microsomes
Native microsomes
Trypsintreated microsomes
26.1 +, 1.65
13.1 +, 0.59
6.37 +, 0.30
4.20 +, 0.16
0.29 ± 0.01
0.20 +, 0.01
24.7 +, 1.54
11.8 + 0.39
6.15 +, 0.43
4.37 +, 0.69
0.27 +, 0.01
0.18 +, 0.02
25.2 +, 1.52
12.3 +, 0.56
6.04 +, 0.21
4.04 +, 0.14
0.31 +, 0.02
0.22 ± 0.0l
26.0 _ 1.35
12.9 +, 0.38
6.29 +, 0.34
4.04 +, 0.39
0.29 +, 0.02
0.20 ± 0.01
24.2 +, 1.58
11.8 +, 0.77
5.59 +, 0.30
4.06 +__0.43
0.31 +, 0.01
0.22 +, 0.01
Unsaturated fatty acids and hepatic drug metabolism
417
Table 2. The mean values of the fatty acid composition in the rat liver microsomes expressed as percentage of the total amount bound to phospholipids. Rats were treated with elaidic or linoleic acids
Control Elaidic (40 mg/kg) Elaidic (200 mg/kg) Linoleic (40 mg/kg) Linoleic (200 mg/kg)
Palm±tic (16:0)
Palmitoleic (16:1)
Stearic (18:0)
Oleic (18:1)
Linoleic (18:2)
Tetraeicosaenoic (20:4)
20.4
4.6
31.5
9.4
15.4
17.9
20.4
1.2
28.6
8.7
18.5
22.7
20.8
3.4
30.9
10.7
16.6
16.9
25.1
5.2
29.7
9.8
15.1
14.9
20.4
0.5
29.6
9.3
18.4
20.8
acid (40mg/kg) or linoleic acid (200mg/kg). The amount of stearic acid decreased slightly when rats were treated with elaidic or linoleic acids. Elaidic acid (40mg/kg) and linoleic acid (200 mg/kg) elevated the amount of microsomal linoleic acid. The amount of tetraeicosaenoic acid (arachidonic acid) increased, when rats were given 40 mg/kg elaidic acid or 200 mg/kg linoleic acid. A decrease in this amount could, however, be detected after giving the rats 40 mg/kg linoleic acid. The activities of microsomal N A D P H cytochrome c reductase, aryl hydrocarbon hydroxylase, p-nitroanisole-O-demethylase and the content of cytochrome P-450 tended to be reduced due to elaidic and linoleic acid treatments of rats (Table 3). The reduction was, however, statistically significant only in the case of aryl hydrocarbon hydroxylase, when rats received linoleic acid 40 mg/kg, and in the case of p-nitroanisole-O-demethylase (specific activity) when rats received li~aoleic acid 200 mg/kg (Table 3). In native microsomes the activity of microsomal
UDPglucuronosyltransferase was affected by elaidic and linoleic acids. Both doses of elaidic acid caused a highly significant reduction in the measurable enzyme activity when calculated on fresh liver basis (Table 4). Linoleic acid also caused a significant reduction in the activity of UDPglucuronosyltransferase, when calculated on fresh liver basis. The effect was, however, slightly weaker than that of elaidic acid (Table 4). The specific activity of UDPglucuronosyltransferase was found to be reduced to some extent in native microsomes when rats received elaidic or linoleic acids. This reduction was, however, significant only in the case of elaidic acid at the dose level of 200 mg/kg (Table 4). As expected from earlier results, trypsin treatment strongly elevated the activity of microsomal UDPglucuronosyltransferase (Table 4). It can also be seen from Table 4 that in trypsin-treated microsomes the activity of UDPglucuronosyltransferase was clearly lowered after treating the rats with elaidic or linoleic acids, In trypsin-treated microsomes unlike in native microsomes, a significant reduction
Table 3. The activities of NADPH cytochrome c reductase (as #moles cytochrome c reduced/min), aryl hydrocarbon hydroxylase (as nmoles hydroxylated benzpyrene formed/min), and p-nitroanisole-O-demethylase (as nmoles p-nitrophenol formed/min) and the cytochrome P-450 content (as nmoles) in rat liver microsomes after treating the rats with elaidic or linoleic acids. Column A represents the activity/g tissue wet weight and column B represents the specific activity. The means and standard errors of the means are expressed. Statistical analysis of the results have been performed with regard to the control group by using Student's t-test. Each group contained five animals NADPH cytochrome c reductase
Control Elaidic (40 mg/kg) Elaidic (200 mg/kg) Linoleic (40 mg/kg) Linoleic (200 mg/kg)
Aryl hydrocarbon hydroxylase
p-nitroanisole-Odemethylase
Cytochrome P-450
A
B
A
B
A
B
A
B
10.3 + 1.6 9.4 ±_ 1.8 10.1 ± 1.1 7.6 + 0.9 9.5 ± 1.8
0.40 + 0.06 0.39 + 0.08 0.41 ± 0.05 0.30 ± 0.04 0.39 ± 0.07
0.69 _+ 0.11 0.60 ± 0.10 0.64 ± 0.10 0.26t ± 0.06 0.56 ± 0.14
0.027 +__0.005 0.024 +_ 0.004 0.025 + 0.004 0.010" + 0.003 0.023 ± 0.005
9.2 + 1.3 8.0 ___0.2 7.0 ± 0.3 7.9 ± 0.4 6.4 ± 0.6
0.40 + 0.05 0.31 ± 0.03 0.28 ± 0.02 0.30 ± 0.01 0.27* ± 0.02
13.2 + 1.1 11.8 ± 1.3 11,3 + 0.6 10,7 ± 1.5 10.9 ± 1.4
0.51 + 0.04 0.48 ± 0.05 0.45 ± 0,02 0.42 ± 0,07 0.45 ± 0.05
* P < 0.05, t P < 0.01.
418
MA'rrl LANG Table 4. The activity of UDPglucuronosyltransferase, as nmoles p-nitrophenol conjugated/min, in rat liver microsomes after treating the rats with elaidic or linoleic acids. Column A represents the activity/g tissue weight and column B represents the specific activity. The means and standard error of the means are expressed: statistical analysis of the results has been performed with regard to the control group by using Student's t-test. Each group contained five animals Native microsomes
Control Elaidic (40 mg/kg) Elaidic (200 mg/kg) Linoleic (40 mg/kg) Linoleic (200 mg/kg)
Trypsin-treated microsomes
A
B
A
B
36.0 ± 1.0
1.35 ± 0.10
161.7 ± 6.9
12.0 + 0.5
28.2 ± 0.8~
1.16 ± 0.07
120.0 ± 6.1t
9.6 ± 0.9*
26.7 ± 1.2~
0.99 ± 0.08*
104.9 ± 4.3~
8.5 ± 0.6?
31.3 ± 1.2"
1.21 ± 0.03
120.6 ± 7.0f
9.4 ± 0.6f
28.3 + 2.0t
1.19 ± 0.11
112.0 ± 10.7~"
10.6 ± 0.3*
* P < 0.05, f P < 0.01, ~ P < 0.001. in the specific activity of UDPglucuronosyltransferase could be measured in every group receiving fatty acids (Table 4). DISCUSSION According to the present results, it seems that unsaturated fatty acids are able to lower the capacity of microsomal drug metabolizing enzymes, both in the mono-oxygenase system and in glucuronidation. In addition to this overall tendency, it can be seen that changes in the enzyme activities caused by fatty acids are not identical. Accordingly, the activity of aryl hydrocarbon hydroxylase is decreased most when linoleic acid (40 mg/kg) is given to rats, p-nitroanisole-Odemethylase activity decreased most when rats received linoleic acid (200 mg/kg) and the greatest decrease in UDPglucuronosyltransferase activity was detected after giving rats elaidic acid (200 mg/kg). Norred & Wade (1972) have found that the catalytic activity of rat liver microsomal mono-oxygenase system increases if a fat-free diet is supplemented with corn oil. In addition, Rowe & Wills (1976) have found that an increase in the unsaturation degree of dietary triglycerides leads to an elevation in the activity of microsomal oxidative demethylation. Similarly, Agradi et al. (1975) have shown that the hydroxylat±on rate of both benzpyrene and aniline is elevated in rat liver microsomes if the average unsaturation degree of the dietary triglycerides is increased. From the reports mentioned above it is, however, difficult to evaluate whether the effect of dietary triglycerides on drug metabolism is due to a certain fatty acid or due to the combined effect of all the fatty acids plus glycerol. When the present results are compared to earlier reports it seems, however, that unsaturated fatty acids act quite differently on microsomal drug metabolism than dietary unsaturated triglycerides. The effect of arachidonic acid (as ethyl arachidonate) and docosa-
hexaenoic acid (as menhaden oil) on microsomal drug metabolism has been studied by Wade et al. (1972). Various doses of acids were given per orally. It was found that both aniline hydroxylase and hexobarbital oxidase activities in microsomes were decreased to various extent depending on the dose (reference group received corn oil instead of fatty acids). This is in agreement with our results, indicating that unsaturated fatty acids are able to decrease the activity of microsomal hydroxylation reactions at least towards some substrates. The question of the possible mechanism by which fatty acids act on the activities of microsomal drug metabolizing enzymes is difficult to answer. According to the results presented here, fatty acids were able to decrease slightly the amount of microsomal phospholipids. It can also be seen that changes in the activities of microsomal p-nitroanisole-O-demethylase and UDPglucuronosyltransferase but not aryl hydrocarbon hydroxylase correlate well with the changes in the amount of microsomal phospholipids. Although the changes in the amount of microsomal phospholipids were not statistically significant they might, however, be enough to cause significant changes in the activities of some drug metabolizing enzymes. On the other hand, it can be seen that elaidic and linoleic acids were able to modify to some extent the fatty acid composition of phospholipid in microsomes. This might also be one factor in regulating the microsomal enzyme activities towards some substrates (for example, benzpyrene). It is interesting to notice that after trypsin treatment, which is presumed to peel off proteins from the outer layers of microsomal membranes, and reveal UDPglucuronosyltransferase (H~inninen & Puukka, 1970), the decrease in the specific activity of UDPglucuronosyltransferase is more evident when compared to the decrease in native microsomes. It should also be noticed that in trypsin-treated microsomes, changes in UDPglucuronosyltransferase activity do not correlate well
Unsaturated fatty acids and hepatic drug metabolism with the changes in the amount of microsomal phospholipids (unlike in the case of native microsomes). This could mean that the catalytic activity of U D P glucuronosyltransferase is highly dependent on the quality of its phospholipid environment. In our earlier studies we have found that dietary cholesterol is able to increase the activities of microsomal drug metabolizing enzymes. The fatty acid composition of microsomal phospholipids is also altered due to dietary cholesterol (Laitinen, in press) and the changes are partly opposite to those caused by elaidic or linoleic acids. Thus dietary cholesterol tended to decrease the amount of microsomal arachidonic and linoleic acids and increase the amount of palmitoleic acid while the dose of 200 mg/kg linoleic acid and 40 mg/kg elaidic acid had an opposite effect on the amount of these fatty acids in microsomes. The results suggest that unsaturated fatty acids may decrease the activity of microsomal drug metabolizing enzymes, partly by lowering the amount of microsoreal phospholipids and partly by modifying the fatty acid composition of microsomal phospholipids. The effects are to some extent opposite to those caused by dietary cholesterol.
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