XENOBIOTICA,
1991, VOL. 21,
NO.
7, 859-864
Effects of simvastatin, a lipoprotein-lowering drug, on the hepatic enzymes involved in drug metabolism in the Wistar rat F. MERCENNEt, H. GOUDONNET, J. MOUNIE, A. ESCOUSSE and R. C. TRUCHOT
Xenobiotica Downloaded from informahealthcare.com by McMaster University on 10/27/14 For personal use only.
Formation de Biochimie Pharmacologique, UFR de Pharmacie et de Medecine, 7 Bd J. D’Arc, 21000 Dijon, France
Received 2 August 1990; accepted 1 January 1991 1. Simvastatin, a competitive inhibitor of 3-hydroxy-3-methyl glutaryl CoA reductase, lowers the plasma cholesterol level and has been approved for treatment of h yperlipoproteinaemia. 2. Simvastatin has been studied for its effects on hepatic microsomal drug metabolism in rat. No induction of 7-ethoxyresorufin-0-deethylase (EROD), ethoxycoumarin-0deethylase (ECOD) and of UDP-glucuronosyltransferases were found, in vitro, after administration of 0.5,1.5 and 10mg/kg per day for 22 days.
3. Epoxide hydrolases (microsomal and cytosolic) were also unchanged after treatment with simvastatin. 4. No increase of the palmitoyl CoA oxidase activity or of mitochondria1 glycerol phosphate dehydrogenase activity occurred. 5. Fatty acid distribution in rat liver microsomal phosphatidylcholines showed a significant decrease of C16:l and a significant increase of C20:4 acids.
Introduction The treatment of hyperlipoproteinaemia is an important factor in the prevention of atheromatosis, a rise in plasmatic cholesterol being considered a major risk factor in the development of cardiovascular disease. Drugs such as resins or fibrates are commonly prescribed to reduce plasmatic cholesterol and lipids. The recent development of specific competitive inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG CoA reductase), such as lovastatin, simvastatin or pravastatin (Tobert 1987, Mol et al. 1988, Schulzeck et al. 1988, Yoshino et al. 1988), offers a new potent therapy for patients with primary hypercholesterolaemia. Fibrates, another class of hypolipidaemic drugs, are known to induce the hepatic microsomal enzymes of the xenobiotic metabolism and the proliferation of peroxisomes in the rat (Schladt et al. 1987, Siest et al. 1978). However, to our knowledge, the effects of simvastatin (figure l), a semisynthetic analogue of compactin (Tobert 1987), on these parameters has not been studied previously. Its effects on phase I and phase I1 enzymes of drug metabolism, and on one of the peroxisomal proliferation markers, palmitoyl CoA oxidase, (Sharma et al. 1988, Lock et al. 1989) were evaluated. Fatty acid distribution of the microsomal lecithins was also analysed, as several microsomal enzymes are highly dependent on their lipidic environment (Siest et al. 1978). f To whom correspondence should be addressed. 0049-8254/91 $3.00 0 1991 Taylor & Francis Ltd.
Xenobiotica Downloaded from informahealthcare.com by McMaster University on 10/27/14 For personal use only.
860
F. Mercenne et al.
Figure 1. Structural formula of simvastatin.
Experimental Animals and treatment Thirty-two male Wistar rats from an SPF husbandry (IFFA Credo, France), weighing 80-100g at the beginning of the experiment, were used. They were kept at a constant temperature (26+1”C) and had free access to tap water and food (Aliment ‘Control’ UAH). The animals were divided into four groups of eight rats: one control group and three groups treated with simvastatin. Simvastatin (Merck-Sharp & Dohme, USA) was solubilized in corn-oil and was administered orally daily for 22 days at 0.5,1.5 and 10 mg/kg per day. T h e same volume of corn-oil was used for all animals (0.33m1/100g of body weight). Preparation of liver subcellular fractions The rats were killed 24h after the last administration of simvastatin, by severing the carotid artery. The liver of each rat was removed, weighed and perfused with ice-cold 0 . 1 5 4 ~KCI solution. A 20% w/v homogenate was prepared with Tris-EDTA buffer (0.02 M Tris, 0.1 M EDTA, 0 , 1 5 4 KCI, ~ p H 7.4) by means of a Potter-Elvehjem Teflon-glass homogenizer. T h e homogenate was successively centrifuged using a Beckman J21-C centrifuge, at 750g for lOmin, at 8700g for 15 min and at 25 OOOg for 15 min. The mitochondrial pellet was collected from the 8700g fraction and the peroxisome-enriched pellet from the 25 OOOg fraction. Both were resuspended in 2.5 ml of the Tris-KCI buffer, but without EDTA, and stored at -80°C. The supernatant was centrifuged at lO5OOOg for 90min using a Beckman L5-50 preparative ultracentrifuge with a 42 1 rotor. T h e microsome fraction was finally resuspended in the Tris-KCI buffer, without EDTA and stored at -80°C. The protein concentration of the subcellular fractions was determined by the method of Lowry et al. (1 951) automatized on a ‘Cobas Bio’ (Roche), a centrifuge autoanalyser, using bovine serum albumin as standard. Analytical methods Microsomal cytochrome P450 content was determined according to Ornura and Sat0 (1964); the ethoxyresorufin-0-deethylase and ethoxycoumarin-0-deethylase activities were measured by the Burke and Mayer (1974) and Greenlee and Poland (1978) methods respectively. UDP-glucuronosyl transferase activities to para-nitrophenol, a planar substrate ( G T I ) , and to NOPOL ((lR)-6,6dimethylbicyclo[3.l.l]hept-2-ene-2-ethanol), a bicyclic monoterpenoid alcohol and a bulky substrate (GT2) were determined by Mulder and Van Doom’s method (1975) automated on a Cobas Bio according to Colin-Neiger et al. (1984). Bilirubin-UDPGT activity was measured by the method of Black et al. (1970). Microsomal and cytosolic epoxide hydrolases were determined according to Dansette et al. (1979) and Oesch and Daly (1 971), respectively, using benzo(a)pyrene-4,5-oxide as substrate for the microsoma1 epoxide hydrolase, and trons-stilbene oxide for the cytosolic one. Assay of mitochondrial glycerol phosphate dehydrogenase was performed using the method of Gardner (1974). Peroxisomal palmitoyl CoA oxidase activity was estimated b y the method of Lazarrow (1981) using the peroxisome-enriched fraction. The lipid fraction of the microsomal suspension was extracted according to Folch et 01. (1957), and phosphatidylcholine was obtained by thin-layer chromatography according to Stahl (1973). T h e methylated derivatives of fatty acids (methylation by NaOH/BF,), were analysed on a Becker-Packard
861
Simvastatin and drug metabotism in the Wistar rat
419 gas chromatograph using a 3 7 m Carbovax 2 0 capillary ~ column. T h e gas chromaography conditions were as follows: nitrogen gas vector, column temp., 192°C; detector temp., 240°C and injector temp., 225°C. A Delsi Enica 21 integrator measured the respective areas of each fatty acid which were expressed in percentage of the total area of all detected fatty acids. Statistics
Results are means k SEM for each group with n = 8 except for glucuronosyl transferases ( G T l and G T 2 ) determinations: six and five rats, respectively, were randomly selected. Means were compared to the control lot by Duncan’s t test. (a) P
1991, VOL. 21,
NO.
7, 859-864
Effects of simvastatin, a lipoprotein-lowering drug, on the hepatic enzymes involved in drug metabolism in the Wistar rat F. MERCENNEt, H. GOUDONNET, J. MOUNIE, A. ESCOUSSE and R. C. TRUCHOT
Xenobiotica Downloaded from informahealthcare.com by McMaster University on 10/27/14 For personal use only.
Formation de Biochimie Pharmacologique, UFR de Pharmacie et de Medecine, 7 Bd J. D’Arc, 21000 Dijon, France
Received 2 August 1990; accepted 1 January 1991 1. Simvastatin, a competitive inhibitor of 3-hydroxy-3-methyl glutaryl CoA reductase, lowers the plasma cholesterol level and has been approved for treatment of h yperlipoproteinaemia. 2. Simvastatin has been studied for its effects on hepatic microsomal drug metabolism in rat. No induction of 7-ethoxyresorufin-0-deethylase (EROD), ethoxycoumarin-0deethylase (ECOD) and of UDP-glucuronosyltransferases were found, in vitro, after administration of 0.5,1.5 and 10mg/kg per day for 22 days.
3. Epoxide hydrolases (microsomal and cytosolic) were also unchanged after treatment with simvastatin. 4. No increase of the palmitoyl CoA oxidase activity or of mitochondria1 glycerol phosphate dehydrogenase activity occurred. 5. Fatty acid distribution in rat liver microsomal phosphatidylcholines showed a significant decrease of C16:l and a significant increase of C20:4 acids.
Introduction The treatment of hyperlipoproteinaemia is an important factor in the prevention of atheromatosis, a rise in plasmatic cholesterol being considered a major risk factor in the development of cardiovascular disease. Drugs such as resins or fibrates are commonly prescribed to reduce plasmatic cholesterol and lipids. The recent development of specific competitive inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG CoA reductase), such as lovastatin, simvastatin or pravastatin (Tobert 1987, Mol et al. 1988, Schulzeck et al. 1988, Yoshino et al. 1988), offers a new potent therapy for patients with primary hypercholesterolaemia. Fibrates, another class of hypolipidaemic drugs, are known to induce the hepatic microsomal enzymes of the xenobiotic metabolism and the proliferation of peroxisomes in the rat (Schladt et al. 1987, Siest et al. 1978). However, to our knowledge, the effects of simvastatin (figure l), a semisynthetic analogue of compactin (Tobert 1987), on these parameters has not been studied previously. Its effects on phase I and phase I1 enzymes of drug metabolism, and on one of the peroxisomal proliferation markers, palmitoyl CoA oxidase, (Sharma et al. 1988, Lock et al. 1989) were evaluated. Fatty acid distribution of the microsomal lecithins was also analysed, as several microsomal enzymes are highly dependent on their lipidic environment (Siest et al. 1978). f To whom correspondence should be addressed. 0049-8254/91 $3.00 0 1991 Taylor & Francis Ltd.
Xenobiotica Downloaded from informahealthcare.com by McMaster University on 10/27/14 For personal use only.
860
F. Mercenne et al.
Figure 1. Structural formula of simvastatin.
Experimental Animals and treatment Thirty-two male Wistar rats from an SPF husbandry (IFFA Credo, France), weighing 80-100g at the beginning of the experiment, were used. They were kept at a constant temperature (26+1”C) and had free access to tap water and food (Aliment ‘Control’ UAH). The animals were divided into four groups of eight rats: one control group and three groups treated with simvastatin. Simvastatin (Merck-Sharp & Dohme, USA) was solubilized in corn-oil and was administered orally daily for 22 days at 0.5,1.5 and 10 mg/kg per day. T h e same volume of corn-oil was used for all animals (0.33m1/100g of body weight). Preparation of liver subcellular fractions The rats were killed 24h after the last administration of simvastatin, by severing the carotid artery. The liver of each rat was removed, weighed and perfused with ice-cold 0 . 1 5 4 ~KCI solution. A 20% w/v homogenate was prepared with Tris-EDTA buffer (0.02 M Tris, 0.1 M EDTA, 0 , 1 5 4 KCI, ~ p H 7.4) by means of a Potter-Elvehjem Teflon-glass homogenizer. T h e homogenate was successively centrifuged using a Beckman J21-C centrifuge, at 750g for lOmin, at 8700g for 15 min and at 25 OOOg for 15 min. The mitochondrial pellet was collected from the 8700g fraction and the peroxisome-enriched pellet from the 25 OOOg fraction. Both were resuspended in 2.5 ml of the Tris-KCI buffer, but without EDTA, and stored at -80°C. The supernatant was centrifuged at lO5OOOg for 90min using a Beckman L5-50 preparative ultracentrifuge with a 42 1 rotor. T h e microsome fraction was finally resuspended in the Tris-KCI buffer, without EDTA and stored at -80°C. The protein concentration of the subcellular fractions was determined by the method of Lowry et al. (1 951) automatized on a ‘Cobas Bio’ (Roche), a centrifuge autoanalyser, using bovine serum albumin as standard. Analytical methods Microsomal cytochrome P450 content was determined according to Ornura and Sat0 (1964); the ethoxyresorufin-0-deethylase and ethoxycoumarin-0-deethylase activities were measured by the Burke and Mayer (1974) and Greenlee and Poland (1978) methods respectively. UDP-glucuronosyl transferase activities to para-nitrophenol, a planar substrate ( G T I ) , and to NOPOL ((lR)-6,6dimethylbicyclo[3.l.l]hept-2-ene-2-ethanol), a bicyclic monoterpenoid alcohol and a bulky substrate (GT2) were determined by Mulder and Van Doom’s method (1975) automated on a Cobas Bio according to Colin-Neiger et al. (1984). Bilirubin-UDPGT activity was measured by the method of Black et al. (1970). Microsomal and cytosolic epoxide hydrolases were determined according to Dansette et al. (1979) and Oesch and Daly (1 971), respectively, using benzo(a)pyrene-4,5-oxide as substrate for the microsoma1 epoxide hydrolase, and trons-stilbene oxide for the cytosolic one. Assay of mitochondrial glycerol phosphate dehydrogenase was performed using the method of Gardner (1974). Peroxisomal palmitoyl CoA oxidase activity was estimated b y the method of Lazarrow (1981) using the peroxisome-enriched fraction. The lipid fraction of the microsomal suspension was extracted according to Folch et 01. (1957), and phosphatidylcholine was obtained by thin-layer chromatography according to Stahl (1973). T h e methylated derivatives of fatty acids (methylation by NaOH/BF,), were analysed on a Becker-Packard
861
Simvastatin and drug metabotism in the Wistar rat
419 gas chromatograph using a 3 7 m Carbovax 2 0 capillary ~ column. T h e gas chromaography conditions were as follows: nitrogen gas vector, column temp., 192°C; detector temp., 240°C and injector temp., 225°C. A Delsi Enica 21 integrator measured the respective areas of each fatty acid which were expressed in percentage of the total area of all detected fatty acids. Statistics
Results are means k SEM for each group with n = 8 except for glucuronosyl transferases ( G T l and G T 2 ) determinations: six and five rats, respectively, were randomly selected. Means were compared to the control lot by Duncan’s t test. (a) P
