Rat Liver Microsomal Activity Research Articles

Mutagenic evaluation of primaquine, pentaquine and pamaquine in the Salmonella/mammalian microsome assay

The 8-aminoquinolines, primaquine, pentaquine and pamaquine, were investigated for mutagenic activity in Salmonella typhimurium strains TA100, TA98, TA97 and TA102 in the rat liver microsomal activation system. Primaquine and pentaquine induced mutations in TA97 in the presence and absence of S9 mix. Pamaquine was mutagenic to TA98 only in the absence of S9 mix.

Effects of oral thalidomide on rat liver and skin microsomal P450 isozyme activities and on urinary porphyrin excretion: interaction with oral hexachlorobenzene.

Adult female Wistar rats (n = 48) divided into four groups of 12 were treated orally with 3 mg/kg per day thalidomide, a 0.05% hexachlorobenzene (HCB)--containing diet, with both drugs together and with the vehicles (controls) over periods of 10 and 60 days. The protein and P450 contents and the activities of aminopyrine-N-demethylase (ADM) and 7-ethoxyresorufin-O-deethylase (7-ERO-D) were determined in the liver microsomes. The activity of 7-ERO-D was also determined in the skin microsomes and total porphyrins were measured in the urine of the animals. Thalidomide increased the hepatic P450 content, caused distinct changes in the activities of the hepatic and cutaneous microsomal isozymes, modified their induction by HCB and inhibited the porphyrogenic activity of HCB. These findings indicate an interaction between thalidomide and HCB with regard to their effects on the P450 isozymes and the metabolism of porphyrins.

In vivo administration of H 2 blockers, cimetidine and ranitidine, reduced the contents of the cytochrome P450IID (CYP2D) subfamily and their activities in rat liver microsomes

1. 1. The effects of in vivo administration of H 2 blockers, cimetidine and ranitidine (0.6 mmol/kg body weight/day, for 5 days), on several P450 isozymes, the P450IID (CYP2D) subfamily, and their monooxygenase activities in rat liver microsomes were investigated. 2. 2. In vivo administration of cimetidine and ranitidine decreased the contents of P450 isozymes and the activities of P450-linked monooxygenase systems; i.e., benzphetamine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarine O-deethylase, debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase. 3. 3. The inhibitory effect on the enzymatic activities of the P450IID (CYP2D)-linked monooxygenase systems was studied by Western blot analysis with serum containing antiCYP2D6 IgG, i.e., LKM1 autoantibody. The amount of P450IID (CYP2D) in liver microsomes decreased more remarkably in the group administered ranitidine or cimetidine in vivo than in controls. 4. 4. The effects of cimetidine and ranitidine on the activities of the P450IID (CYP2D)-linked monooxygenase systems were investigated in vitro. The activities of debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase were inhibited in vitro by cimetidine or ranitidine at a higher concentration than that on in vivo administration of either H 2 blocker. 5. 5. The kinetic parameters for cimetidine or ranitidine as to the activities of debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase in liver microsomes were determined by means of Lineweaver-Burk plots. 6. 6. The suppressive effects of cimetidine and ranitidine on the activities of the P450IID (CYP2D)-linked monooxygenase systems in vivo were found to be due to a decrease of the content of the P450IID (CYP2D) protein.

Effect of chlorinated dibenzo-p-dioxins on 7-ethoxycoumarin O-deethylase activity in rat liver microsomes

The effect of dibenzo- p-dioxin (DD) and chlorinated dibenzo- p-dioxins (CDDs) on the cytochrome P450 (P450)-dependent monooxygenase, 7-ethoxycoumarin O-deethylase (ECOD) in rat liver microsomes was examined in vitro. ECOD activity was induced 21.9- and 5.8-fold over that of the control by 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreatment, respectively. Moreover, anti-P450IA1 immunoglobulin G (IgG) inhibited ECOD activity in liver microsomes of rats pretreated with MC, but was not in that pretreated with PB. ECOD activities in MC and PB-pretreated rat liver microsomes in the presence of 100 μM DD or CDDs were inhibited to 18–70% and 31–91% of the control activity, respectively. In the liver microsomes of rats pretreated with MC, the inhibition constant (Ki) for 1,2,4-tetrachlorodibenzo- p-dioxin (1,2,4-TrCDD) was the lowest (5.41 μM) among the compounds tested, which was the same as that using β-naphthoflavone as the positive control, followed by 2-monochlorodibenzo- p-dioxin (2-MCDD). On the other hand, in PB-pretreated rat liver microsomes, the apparent Ki values were 25.9 and 156 μM for 2-MCDD and 1,2,4-TrCDD, respectively. Thus, 1,2,4-TrCDD strongly inhibited ECOD activity in MC-pretreated rat liver microsomes, and 2-MCDD inhibited it in those of both MC- and PB-pretreated rats. Therefore, in vitro, DD and CDDs could be classified into two groups: a MC-type such as 1,2,4-TrCDD and a mixture type of MC and PB such as 2-MCDD. Our results suggested that the inhibition profile of ECOD by dioxin compounds in vitro is useful for predicting their toxicity.

Induction of coenzyme A-dependent transacylation activity in rat liver microsomes by administration of clofibrate

The effect of administration of clofibrate on the activity of coenzyme A-dependent (CoA-dependent) transacylation of 1-acyl-glycerophosphocholine (1-acyl-GPC) was examined in rat liver microsomes. Administration of clofibrate to rats increased the activity of CoA-dependent transacylation of 1-[ 14C]acyl-GPC and the activity reached a value (8.37 nmol/min per mg protein) twice that in control rats (3.95 nmol/min per mg protein) without any changes in apparent K m values for CoA (1.2 μM in control and 1.0 μM in clofibrate-treated) and 1-acyl-GPC (33.4 μM in control and 27.8 μM in clofibrate-treated). The rate of CoA-dependent transfer of [ 14C]arachidonic acid (20:4) from 1-acyl-2-[ 14C]20:4-glycerophosphoethanolamine (GPE) or 1-acyl-2-[ 14C]20:4-glycerophosphoinositol (GPI) to 1-acyl-GPC (synthesis of 1-acyl-2-[ 14C]20:4-GPC) was also increased by treatment with clofibrate (1.9-fold and 1.5-fold increases, respectively). These results suggest that a CoA-dependent transacylation system of 1-acyl-GPC was induced by treatment with clofibrate.

Purification of diacylglycerol:acyltransferase from rat liver to near homogeneity.

A method to isolate a protein related to the diacylglycerol:acyltransferase (DGAT) activity in rat liver microsomes has been developed. The microsomes were treated with sodium deoxycholate (DOC; 0.1 mg/mg protein) at a concentration of 1 mM, i.e., below the critical micellar concentration (CMC), to remove luminal and loosely bound proteins. Three percent of the DGAT activity and all of the acylCoA hydrolyse activity were present in the supernatant, i.e., among the extracted loosely bound proteins. The insoluble material, recovered as a pellet, was suspended in DOC (1.6 mg/ml and mg protein in the original microsomes), and subjected to multiple, short (1-2 sec) sonications. CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; 5 mg/ml) was then added, and the sonication was repeated. The detergent-treated microsomal membranes were filtered through a 0.22-micron filter and chromatographed on a Superose 6 column from which the DGAT activity was recovered in a high molecular mass fraction. A monoclonal antibody that reacted with this fraction was raised and used in immunoaffinity experiments. This antibody removed 93 +/- 6% (mean +/- SD, n = 4) of the DGAT activity present in solution and 44 +/- 6% (mean +/- SD, n = 5) of the applied activity could be recovered after desorption. The antibody recognized a 60 kDa protein upon Western blot of rat liver microsomal proteins as well as of the DGAT-containing fraction from the Superose 6 column. A 60 kDa protein was highly enriched in the DGAT-containing retained fraction from the immunoaffinity chromatography. This 60 kDa protein reacted with the monoclonal antibody on Western blot. In addition to the 60 kDa protein, the retained fraction from the immunoadsorber contained a 77 kDa protein. This protein did not react with the monoclonal antibody on Western blots. Neither the 60 nor the 77 kDa protein reacted with antibodies to mouse immunoglobulins or showed any unspecific reaction with immunoglobulins.

Effect of aromatase inhibitors on estrogen 2-hydroxylase in rat liver

The effect of aromatase inhibitors, 4-hydroxyandrostenedione, CGS 16949A and aminoglutethimide on the inhibition of estrogen 2-hydroxylase activity in rat liver microsomes in vitro and on its induction in vivo has been examined. Estrogen 2-hydroxylase was found to have over twice the affinity for estradiol compared to estrone. Using high pressure liquid chromatography and employing estradiol as a substrate, the IC 50 values were 2.2, 98, 110 and 908 μM for the reference compound ketoconazole and the aromatase inhibitors, 4-hydroxyandrostenedione, CGS 16949A and aminoglutethimide, respectively. Similar IC 50 values were obtained using estrone as a substrate and by a tritiated water method employing estradiol as a substrate. The K m value for estrogen 2-hydroxylase with estradiol as a substrate using a tritiated water method was 4.3 μM with a V max of 11.89 nmol/h/mg. Ketoconazole, CGS 16949A and aminoglutethimide exhibited non-competitive inhibition whereas 4-hydroxyandrostenedione appeared to be a competitive inhibitor of estrogen 2-hydroxylase. The K i values were 2.6, 72, 114 and 958 μM for ketoconazole, 4-hydroxyandrostenedione, CGS 16949A and aminoglutethimide, respectively. All three aromatase inhibitors were weak inhibitors of estrogen 2-hydroxylase as compared to the reference drug, ketoconazole. Following treatment of rats with aminoglutethimide (40 mg/kg/day; i.p.; for 3 days), estrogen 2-hydroxylase activity was increased by 28 and 30% using estradiol and estrone as substrates, respectively. Following treatment of rats with CGS 16949A (2 mg/kg/day; p.o.; for 3 days), the corresponding increase in estrogen 2-hydroxylase activity was 48 and 44%. The results of this study indicate that the aromatase inhibitors, aminoglutethimide and CGS 16949A re only weak inhibitors of estrogen 2-hydroxylase activity in vitro and show no evidence of inhibition in vivo. On the contrary, there was some evidence to suggest that both aminoglutethimide and CGS 16949A induce estrogen metabolism following repeated administration. Therefore, aminoglutethimide and CGS 16949A may lower estrogen levels not only by primarily inhibiting their synthesis but also by inducing the metabolism of estrogens.

Metabolism of FK506, a potent immunosuppressive agent, by cytochrome P450 3A enzymes in rat, dog and human liver microsomes

The oxidative metabolism of FK506 by liver microsomes and purified cytochrome P450 (P450) enzymes from rats, dogs and humans was studied. The major metabolite formed by liver microsomes from all species was 13-demethylated FK506, named M-I. In adult rats, liver microsomal metabolic activity toward FK506 was higher in males than in females and was stimulated by treatment with P450 3A inducers such as dexamethasone and phenobarbital. In a reconstituted monooxygenase system containing various forms of purified P450 3A enzymes, rat P450 3A2, dog P450 DPB-1 (a form of the P450 3A family) and human P450 3A4 catalyzed FK506 oxidation efficiently in the presence of cytochrome b 5, a mixture of phospholipids (dilauroylphosphatidylcholine, dioleoylphosphatidylcholine and phosphatidylserine), and sodium cholate. Rat P450 2C6 and 2D1 and human P450 2C MP also metabolized FK506, with significant lower activity than the P450 3A enzymes, and other rat P450 1A, 2A, 2B, 2C and 2E families including C11 did not show catalytic activities for FK506. Anti-P450 3A2 and anti-P450 3A4 antibodies strongly inhibited FK506 oxidation catalyzed by rat and human liver microsomes, respectively. The formation rate of M-I correlated well with testosterone 2β- and 6β-hydroxylase activities in rat liver microsomes and with immunoquantified P450 3A4 content, nifedipine oxidase activity, and testosterone 6β-hydroxylase activity in human liver microsomes. These in vitro findings indicate that the P450 3A enzymes in liver microsomes from various species of animals, including human, play a major role in the first step oxidation of FK506.

Synthesis and anti lipid-peroxidation activity of hydroquinone monoalkyl ethers.

A series of hydroquinone monoalkyl ethers was synthesized and evaluated for anti lipid-peroxidation activity in rat liver microsomes. 4-Hexyloxy-2,3,6-trimethylphenol (9), having a low redox potential, as well as ascorbic acid exhibited the strongest anti lipid-peroxidation activity (IC50 = 4.2 x 10(-7) M). Structure-activity relationship studies demonstrated that the inhibitory effect of hydroquinone monoalkyl ethers on lipid peroxidation was increased by the acquisition of an optimum hydrophobicity and decreased by an insufficient or excessive hydrophobicity.

Identification of N-acetylcysteine as a new substrate for rat liver microsomal glutathione transferase. A study of thiol ligands.

N-Acetyl-L-cysteine serves as an efficient substrate for the rat liver microsomal glutathione transferase with 1-chloro-2,4-dinitrobenzene as second substrate (8.8 +/- 0.37 mumol/min mg). The activity is actually higher than that obtained with glutathione (2-4 mumol/min mg). In examining the activity of liver subcellular fractions, no activity with N-acetyl-L-Cys could be detected in dialyzed or N-ethylmaleimide-treated (in order to remove endogenous glutathione) cytosol. The activity in rat liver microsomes was 0.11 +/- 0.007 mumol/min mg, which is accounted for by the content of microsomal glutathione transferase. Thus, N-acetyl-L-Cys can be used as a specific substrate for determining the conjugating activity of microsomal glutathione transferase. N-Acetyl-L-Cys was also shown to function as a substrate for the enzyme when other second substrates than 1-chloro-2,4-dinitrobenzene (with varying electrophilicity) are used. The pH dependence of microsomal glutathione transferase was studied. The kcat/Km(1-chloro-2,4-dinitrobenzene) was dependent on pH with an apparent pKa of 6, > or = 9, and > or = 8 with saturating glutathione, gamma-L-Glu-L-Cys, and N-acetyl-L- cysteine, respectively. Apparently the enzyme has the ability to lower the pKa of glutathione by 3 orders of magnitude. The kcat/Km(thiol) did not vary appreciably with pH (except for N-acetyl-L-cysteine), indicating that no rate-determining deprotonation occurs on the enzyme itself between pH 5.5 and 9. The abilities of histidine-, lysine-, and arginine-selective reagents to inactivate the enzyme when N-acetyl-L-cysteine and gamma-L-Glu-L-Cys were used as substrates were investigated. The activity toward N-acetyl-L-cysteine was decreased considerably less after treatment with the arginine-selective reagent phenylglyoxal, as compared to the activity toward GSH and gamma-L-Glu-L-Cys. This indicates that an arginine makes contact with gamma-L-Glu residue in GSH. With the other reagent/substrate combinations tested the enzyme was inactivated almost completely. The ability of microsomal glutathione transferase to stabilize the Meisenheimer complex formation between 1,3,5-trinitrobenzene and various glutathione analogues, including non-substrate thiols, has been examined. It is shown that, in general, substrates exhibited higher formation constants (approaching 50 mM-1) than non-substrates (4.5 +/- 1.7 mM-1, n = 7), whereas simpler thiols did not yield enzyme-bound complexes. The fact that the enzyme can stabilize Meisenheimer complexes from non-substrate thiol analogues of glutathione offers new possibilities for examining the substrate interactions of glutathione transferases.

Correspondence of increased debrisoquine 4-monooxygenase activity with seizure-susceptibility in Mongolian gerbils

Four drug-metabolizing activities in Wistar rat and Mongolian gerbil liver microsomes were investigated to clarify the biochemical basis of convulsions. No significant associations were observed between the susceptibility to the tonic-clonic seizures in Mongolian gerbils and the drug-metabolizing enzymatic activities of p-nitroanisole O-demethylase, aminopyrine demethylase and benzo[a]pyrene-3-monooxygenase. However, a significant association was observed with the debrisoquine 4-monooxygenase (cytochrome P-450db1-linked monooxygenase system) activity, which is known to metabolize exogenous and endogenous parkinsonism-inducing neurotoxins such as MPTP and TIQ, and to activate carcinogens during the detoxication of xenobiotics. The mean activity of debrisoquine 4-monooxygenase in the seizure-sensitive group (male, 380; female, 350 pmol/h/mg protein) remained significantly higher (about 4–5 times) than that in the seizure-resistant group (male, 90; female, 80 pmol/h/mg protein). More Mongolian gerbils were extensive metabolizers of debrisoquine in the seizure-sensitive group than in the seizure-resistant group. These results suggest a possibility that the cytochrome P-450db1-linked monooxygenase system may activate the potential neurotoxins which are associated with tonic-clonic seizures in the Mongolian gerbil.

DNA adduct formation by tamoxifen with rat and human liver microsomal activation systems.

Using microsomal preparations from rat and human liver, we investigated the activation of the anti-estrogen compound tamoxifen (TMX) to form DNA adducts. Pretreatment of rats with phenobarbital increased DNA adduct formation by microsomal activation of TMX 3- to 6-fold, depending on the cofactors used. When reduced nicotinamide-adenine dinucleotide phosphate (NADPH) was used as a cofactor in human and rat microsomal activation systems, the relative DNA adduct levels were 2.9 and 5.2 x 10(-8) respectively and 1-3 TMX-DNA adducts were detected by 32P-postlabeling; DNA adduct 1 was the same in both microsomal systems. When cumene hydroperoxide (CuOOH) was used as a cofactor, activation of TMX produced four major DNA adducts and several minor DNA adducts in both rat and human liver microsomes; the relative adduct levels were 11.1 and 23.1 x 10(-8) respectively. TMX-DNA adducts 1, 4, 5 and 6 were similar in both human and rat microsomal systems with CuOOH as the cofactor. The TMX-DNA adducts formed with NADPH as the cofactor were clearly different from those formed with CuOOH as the cofactor, which implies that the metabolites leading to the individual DNA adducts were different. Addition of a P450 inhibitor, either n-octylamine or alpha-naphthylisothiocyanate, to the activation system reduced adduct formation by 70-93%. We propose that the TMX-DNA adducts formed with NADPH as the cofactor result from P450 acting as a mono-oxygenase, whereas the adducts formed with CuOOH as the cofactor result from P450 acting as a peroxidase. Our findings suggest that further studies may be required to establish the safety of TMX treatment of women for purposes other than chemotherapy.

Effects of Oral Administration of Acitretin on Rat Liver Microsomal Phospholipids, P-450 Content and Monooxygenase Activities

Doses of 3 and 10 mg/kg/day acitretin were orally administered to female Wistar rats over a period of 6 weeks. Phospholipid classes, P-450 content, aminopyrine-N-demethylase (ADM) and 7-ethoxyresorufin-O-deethylase (7-ERO-D) activities were determined in the liver microsomes of the treated animals. Both dosages caused statistically significant alterations in rat liver microsomal phospholipid composition which may be associated with changes in the metabolic activity, ionic transport, cell-cell interaction and other processes of the hepatic cellular components. Furthermore, statistically significant alterations of P-450 isozyme activities were induced by both dosages. Our data suggest a possible interaction of acitretin with other drugs and endogenous substances metabolized by these enzyme systems.

Gypsetin, a new inhibitor of acyl-CoA:cholesterol acyltransferase produced by Nannizzia gypsea var. incurvata IFO 9228. I. Fermentation, isolation, physico-chemical properties and biological activity.

A novel inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), designated gypsetin, was isolated from the cultured broth of Nannizzia gypsea var. incurvata IFO 9228 by solvent extraction, silica gel chromatography and crystallization. Gypsetin inhibited rat liver microsomal ACAT activity competitively with respect to the substrate oleoyl-CoA with an apparent Ki value of 5.5 microM. In cultured macrophage J774 cells incubated with oxidized low density lipoprotein, gypsetin inhibited cholesteryl ester formation from [14C]oleate by 50% at a concentration of 0.65 microM without affecting cell surface binding, uptake and degradation of the lipoprotein.

Antimutagenic Activity of Water Extracts of Black Tea and Oolong Tea

Water extracts of leaves of black tea, oolong tea, and green tea, were examined for antimutagenic activity with Salmonella typhimurium test strains, TA 98 and TA 100. These water extracts significantly decreased the reverse mutation induced by crude dimethyl sulfoxide extracts of grilled beef and, by Trp-P-1, Glu-P-1, and B[a]P in the presence of a rat liver microsomal activation system. They also decreased the mutagenicity of AF-2 and 4-NQO requiring no metabolic activation to cause the mutation. Removal of caffeine from tea extracts did not influence the DEGB-induced mutation, but ethyl acetate extraction abolished the effect.

Basal Mg(2+)-dependent ATPase activity of rat liver microsomes is not influenced by ambient free Ca2+.

The potent inhibitor of Ca2+, Mg(2+)-ATPase activity, thapsigargin, has been used to investigate the effect of ambient free Ca2+ on basal Mg(2+)-dependent ATPase activity in rat liver microsomes. Thapsigargin non-competitively inhibited both Ca(2+)-stimulated ATP-dependent Ca2+ uptake and Ca(2+)-stimulated Mg(2+)-dependent ATPase activity. At a concentration of 1 microM, thapsigargin completely inhibited the Ca(2+)-pump activity, without affecting Mg(2+)-dependent ATPase activity measured in the absence of Ca2+. Increasing the ambient free Ca2+ concentration did not alter the basal Mg(2+)-dependent ATPase activity. The data presented indicate that ATPase activity measured in the absence of Ca2+ is a reliable measure for the basal Mg(2+)-dependent ATPase activity and that, consequently, the Ca(2+)-stimulated Mg(2+)-dependent ATPase activity can indeed be defined as the difference between the ATPase activity measured in the presence and the absence of Ca2+.

Determination of p-Nitrophenol Hydroxylase Activity of Rat Liver Microsomes by High-Pressure Liquid Chromatography

p-Nitrophenol hydroxylation to p-nitrocatechol is a useful metabolic marker for the presence of functional cytochrome P450 2E1 in mammalian cell microsomes, but the assay is limited by the sensitivity of the spectrophotometric method used to monitor p-nitrocatechol formation. In this paper, a reverse-phase high-pressure liquid chromatography method, which is nearly 20 times more sensitive than the spectrophotometric method and more specific for p-nitrocatechol determination, is described. The method involves monitoring the presence of p-nitrocatechol in the trifluoroacetic acid-quenched reaction mixtures at 345 am. The utility of the method was demonstrated with rat liver microsomes, where p-nitrocatechol formation was found to be NADPH dependent, was linear with incubation times (2.5 to 30.0 min) and protein concentrations (0.03-0.48 mg/incubation), and exhibited typical Michaelis-Menton kinetics ( K m = 197 μM, V max = 2.8 nmol/mg protein/min).

Participation of P450 3A Enzymes in Rat Hepatic Microsomal Retinoic Acid 4-Hydroxylation

Cytochrome P450-mediated 4-hydroxylation is an important pathway in the termination of the biological action of retinoids. Several purified mammalian hepatic P450s have been shown to catalyze the 4-hydroxylation of all- trans-retinoic acid (retinoic acid) in reconstituted enzyme systems, but the nature of the activity in untreated rat liver microsomes has not been defined. In the present study, microsomal retinoic acid 4-hydroxylation was characterized in untreated liver from rats of both sexes and after a series of induction treatments. Thus, dexamethasone and phenobarbital, but not β-naphthoflavone or dimethyl sulfoxide, increased the activity in male and female rats. An immunoglobulin G (IgG) fraction raised against P450 3A1 decreased the rate of retinoic acid 4-hydroxylation in untreated rat hepatic microsomes, but IgG directed against the P450s 2C11, 2B1, and 2C6 were noninhibitory. In vivo administration of triacetyloleandomycin, which has been shown to form a metabolite intermediate complex with P450 3A, followed by potassium ferricyanide oxidation in vitro, reactivated retinoic acid 4-hydroxylation and androst-4-ene-3,17-dione 6β-hydroxylation similarly (to 154 and 152% of the respective activities in the absence of potassium ferricyanide). Finally, exogenous retinoic acid (60 mg/kg ip for three days) markedly increased the rate of retinoic acid 4-hydroxylation in hepatic microsomes from male and female rats (3.8- and 3.7-fold, respectively). This occurred without comparable increases in other P450 activities. Despite these findings, the age- and sex-related profiles of microsomal retinoic acid 4-hydroxylation were clearly different from those measured for other P450 activities. Thus, on the basis of xenobiotic pretreatment, developmental, and immunochemical studies, the enzyme(s) involved in constitutive retinoic acid 4-hydroxylation appears distinct from a number of well-described P450s in untreated and induced rat liver. The data are consistent with the partial involvement of P450(s) from the 3A subfamily in retinoic acid 4-hydroxylation, but it seems clear that P450s 3A1 and 3A2 do not participate in this activity.

Cholesterol Content of the Rat Lens is Lowered by Administration of Simvastatin, but not by Pravastatin

The influence of the 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors pravastatin and simvastatin on lens cholesterol metabolism was investigated in the rat. Short-term organ culture experiments with explanted lenses from 21-day-old Wistar rats showed that simvastatin was at least 35 times more effective than pravastatin in inhibiting cholesterol synthesis. In vivo the cholesterol content of the rat lens increased linearly with age. Experiments were designed to answer the question whether simvastatin and pravastatin inhibit lens cholesterol synthesis in vivo, which would result in a reduced cholesterol accumulation in the lens with age. Young Wistar rats were weaned at an age of 21 days and had ad libitum access to a chow supplemented with 10-100 mg vastatin kg -1 (drug consumption: 1·5-15 mg vastatin kg -1 body weight day -1, respectively) or no additions for 3 weeks. Both drugs induced the HMG-CoA reductase activity in rat liver microsomes (isolated after 1, 2 and 3 weeks of treatment) to a similar extent. This indicates that the two drugs inhibited hepatic cholesterol synthesis to a comparable extent. During the whole treatment period no significant differences between control and drug-treated animals could be observed when the wet weight and protein content of the lenses were considered. However, a striking difference between the control group and pravastatin group (50 mg drug kg -1 diet) on the one hand and the simvastatin group (50 mg drug kg -1 diet) on the other was observed when the cholesterol content of the lenses were compared as a function of age. After 1 week of treatment all three groups showed the same increase in cholesterol content. Thereafter, the simvastatin group showed little additional increase, whereas the pravastatin group and the control group showed the same increase in cholesterol content (from 20 to 40μg per lens). Even at a concentration of 100 mg kg -1 chow, pravastatin had no effect after 3 weeks, whereas at this concentration simvastatin already caused a reduction in lens cholesterol content after 7 days of treatment. Simvastatin in a concentration of 10 mg kg -1 chow reduced lens cholesterol by more than 25% after 3 weeks. So, we observed at least a ten-fold difference between both drugs in the ability to affect the cholesterol content of the lens in vivo. Furthermore, our observations indicate that in the avascular lens the accumulation of cholesterol with age is largely dependent on in situ de novo synthesis, and that under these in vivo conditions simvastatin, but not pravastatin, inhibits cholesterol synthesis in the lens.

Age-related changes in delta 6 and delta 5 desaturase activities in rat liver microsomes.

Age-related changes in delta 6 desaturation of [1-14C]alpha-linolenic acid and [1-14C]linoleic acid and in delta 5 desaturation of [2-14C]dihomo-gamma-linolenic acid were studied in liver microsomes from Wistar male rats at various ages ranging from 1.5 to 24 mon. Desaturase activities were expressed both as specific activity of liver microsomes and as the capacity of whole liver to desaturate by taking into account the total amount of liver microsomal protein. delta 6 Desaturation of alpha-linolenic acid increased from 1.5 to 3 mon and then decreased linearly up to 24 mon to reach the same desaturation capacity of liver measured at 1.5 mon. The capacity of liver to desaturate linoleic acid increased up to 6 mon and then remained constant, whereas microsomal specific activity was equal at 1.5 and 24 mon of age. The capacity of liver to convert dihomo-gamma-linolenic acid to arachidonic acid by delta 5 desaturation decreased markedly from 1.5 to 3 mon. It then increased to reach, at 24 mon, the same level as that observed at 1.5 mon. Age-related changes in the fatty acid composition of liver microsomal phospholipids at the seven time points studied and of erythrocyte lipids at 1.5 and 24 mon were consistent with the variations in desaturation capacity of liver. In particular, arachidonic acid content in old rats was slightly higher than in young rats whereas contents in linoleic and docosahexaenoic acids varied little throughout the life span.(ABSTRACT TRUNCATED AT 250 WORDS)