Livers Of Clofibrate-treated Rats Research Articles

In VitroMetabolism of Chlorotriazines: Characterization of Simazine, Atrazine, and Propazine Metabolism Using Liver Microsomes from Rats Treated with Various Cytochrome P450 Inducers

Thein vitrometabolism of chlorotriazines, simazine (SIZ), atrazine (ATZ), and propazine (PRZ) was studied using control, 3-methylcholanthrene-, phenobarbital-, pyridine-, dexamethasone-, and clofibrate-treated rat liver microsomes. The metabolites were determined by HPLC. The principal reactions by cytochrome P450 (P450) system wereN-monodealkylation and isopropylhydroxylation in all rat liver microsomes. As a result, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (M1) (SIZ-M1 for SIZ and ATZ-M1 for ATZ) and 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine (M2) (ATZ-M2 for ATZ and PRZ-M2 for PRZ), 2-chloro-4-ethylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazine (M3) (ATZ-M3 for ATZ), and 2-chloro-4-isopropylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazine (M4) (PRZ-M4 for PRZ) were detected as the metabolites.N-bidealkylation and 2-hydroxylation were not found in this system. The formation rates of SIZ-M1, ATZ-M1, ATZ-M2, and PRZ-M2 were markedly induced by 3-methylcholanthrene, phenobarbital, and pyridine. On the other hand, the formation rates of ATZ-M3 and PRZ-M4 were significantly induced by phenobarbital, pyridine, and/or clofibrate, but not by 3-methylcholanthrene. The enzyme kinetics of chlorotriazine metabolism were examined by mean of Eadie–Hofstee analyses. Although there was no remarkable difference ofKmfor the products in chlorotriazine metabolism among the microsomes tested, theVmaxandClint(Vmax/Km) for the products in chlorotriazine metabolism are affected by P450 inducers, except for dexamethasone. The formation rates of SIZ-M1, ATZ-M1, ATZ-M2, and PRZ-M2 were significantly correlated with 7-ethoxyresorufinO-deethylase, acetanilide 4-hydroxylase, 7-ethoxycoumarinO-deethylase, 4-nitrophenol 2-hydroxylase, and testosterone 7α-hydroxylase activities and CYP1A1/2 level, whereas the formation rates of ATZ-M3 and PRZ-M4 were significantly correlated with testosterone 16β-hydroxylase, bufuralol 1′-hydroxylase, and 4-nitrophenol 2-hydroxylase activities and CYP2B1/2 level. These results suggest that the inducibility in metabolism of SIZ, ATZ, and PRZ is different betweenN-monodealkylation and isopropylhydroxylation and that theN-monodealkylation and isopropylhydroxylation are induced by CYP1A1/2, CYP2B1/2, and CYP2B1/2, respectively.

Effect of the side-chain structure on the specificity of beta-oxidation in bile acid biosynthesis in rat liver homogenates

3Alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid (C27-5beta-cholestanoic acid) derivatives with different carbon-number side chains were incubated with rat liver 800 g supernatant to study the effect of the side-chain length on the beta-oxidation system in bile acid biosynthesis. The intermediate alpha, beta-unsaturated and beta-hydroxylated bile acids, and the corresponding degradation products, were quantitatively determined by gas chromatography. The longer side-chained derivatives (C28- and C29-5beta-cholestanoic acids) were converted into corresponding sidechain degradation products, and the alpha,beta-unsaturated and beta-hydroxylated intermediates were also produced. On the other hand, the shorter side-chained derivative (C26-5beta-cholestanoic acid) only gave alpha,beta-unsaturated intermediate. The total formation of intermediates and degradation products from corresponding substrates was in the order of C27- > C28- > C29- > C26-5beta-cholestanoic acids. In the case of clofibrate-treated rat liver 800 g supernatant, the formation of intermediates and final degradation products from C28- and C29-5beta-cholestanoic acids increased significantly. These longer side-chained analogues seemed to be subjected to beta-oxidation system(s) induced with clofibrate treatment. The effect of a terminal methyl group in the side chain of 5beta-cholestanoic acid on the oxidation system was also investigated using 3alpha, 7alpha, 12alpha-trihydroxy-27-nor-5beta-cholestanoic acid derivatives as enzymatic substrates. These derivatives gave corresponding side chain degradation products, but the formation of intermediates was not detected. The formation of side chain cleavage products from 27-nor-5beta-cholestanoic acid derivatives increased to 10 to 25-fold that of the controls by treatment with clofibrate. The results suggested that the beta-oxidation system for 27-nor-5beta-cholestanoic acid derivatives was different from that for C27-5beta-cholestanoic acid, despite their bile acid steroidal structure.

A Sandwich Enzyme-linked Immunosorbent Assay for .DELTA.3, .DELTA.2-Enoyl-CoA Isomerase in Rat-Tissue Homogenates.

We established two monoclonal antibodies (MAbs, A6-1-E4, 1gM; κ and BC-D1, IgG1, λ), that specifically recognize different epitopes of rat Δ3, Δ2-enoyl-CoA isomerase (ECI). We then developed a specific and reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using a capture monoclonal antibody and an indicator polyclonal antibody to evaluate ECI. The amounts of ECI were linearly determined in the ranges from 15 to 250 ng/well by this method. This assay can detect the amount of ECI in tissue homogenates without subfractionation. The ECI levels were 0.13 ng/μg homogenate in normal rat liver and 1.50 ng/μg homogenate in clofibrate-treated rat liver.

Peroxisomal degradation of leukotrienes by beta-oxidation from the omega-end.

Chain shortening via beta-oxidation from the omega-end has been recognized as the major pathway for the degradation of cysteinyl leukotrienes as well as leukotriene B4 (LTB4). The metabolic compartmentation of this pathway was studied using peroxisomes purified from normal and clofibrate-treated rat liver. beta-Oxidation products of omega-carboxy-LTB4, including omega-carboxy-dinor-LTB4 identified by gas chromatography-mass spectrometry, were formed by the isolated peroxisomes. The reaction was dependent on CoA, ATP, and NAD and was stimulated by FAD. NADPH was necessary for the further metabolism of omega-carboxy-dinor-LTB4. Together with microsomes a degradation of omega-carboxy-LTB4 also proceeded in isolated mitochondria in the presence of CoA, ATP, and carnitine. beta-Oxidation of the cysteinyl leukotriene omega-carboxy-N-acetyl-leukotriene E4 was observed only with isolated peroxisomes in combination with lipid-depleted microsomes. Direct photoaffinity labeling using omega-carboxy-[3H] LTB4 and omega-carboxy-N-[3H]acetyl-LTE4 served to identify peroxisomal leukotriene-binding proteins. The bifunctional protein (EC 4.2.1.17 and 1.1.1.35) and 3-ketoacyl-CoA thiolase (EC 2.3.1.16) of the peroxisomal beta-oxidation system were the predominantly labeled polypeptides as revealed by precipitation with monospecific antibodies. In vivo studies with N-acetyl-[3H2]LTE4, N-acetyl-[3H8]LTE4, and N-[14C]acetyl-LTE4 after treatment with the peroxisome proliferator clofibrate indicated formation and biliary excretion of large amounts of metabolites more polar than omega-carboxy-tetranor-N-acetyl-LTE3 including omega-carboxy-tetranor-delta 13-N-acetyl-LTE4 and omega-carboxy-hexanor-N-acetyl-LTE3. Increased formation of beta-oxidized catabolites of N-acetyl-LTE4 and LTB4 was also observed in hepatocytes isolated after clofibrate treatment. Our results indicate that peroxisomes play a major role in the beta-oxidation of leukotrienes from the omega-end. Whereas omega-carboxy-LTB4 was beta-oxidized both in isolated peroxisomes and mitochondria, the cysteinyl leukotriene omega-carboxy-N-acetyl-LTE4 was exclusively degraded in peroxisomes.

Co-suppression by Nicardipine, a Calcium Antagonist, of Induction of Microsomal LauricAcid Hydroxylation with Peroxisome Proliferation in Clofibrate-Treated Rat Liver.

The in vivo effect of nicardipine, a well-known calcium antagonist, on microsomal omega-oxidation of laurate in clofibrate-treated rat liver was studied. The 15.3-fold induction of the activity by 2 weeks administration of 0.25% clofibrate in the diet was markedly suppressed to about 6-fold by co-administration of nicardipine at 100 mg/kg body weight. Similarly, the induction of peroxisomal beta-oxidation and carnitine acetyltransferase activities were also suppressed by this simultaneous administration by more than 50%. Although clofibrate also induced the activity of reduced nicotineamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase and increased the hepatic content of cytochrome P-450, no suppressive effect of nicardipine was observed. Contrarily, nicardipine induced the reductase activity and increased the hepatic content of cytochromes P-450 and b5. These results provide the first demonstration of a calcium antagonist, e.g. nicardipine acting as inhibitor of the induction of microsomal omega-oxidation, in association with the inhibition of peroxisome proliferation in animals. The suppression of drug-induced peroxisome proliferation and microsomal omega-oxidation by the calcium antagonist may help in elucidating the causal relationship of the induction mechanisms between peroxisomal and microsomal enzymes.

Participation of the peroxisomal β-oxidation system in the chain-shortening of PCA 16, a metabolite of the cytosine arabinoside prodrug, YNKO1, in rat liver

When PCA 16, a metabolite of the cytosine arabinoside prodrug YNKO1, was incubated with isolated rat hepatocytes, time-dependent H 2O 2 generation was found. When the hepatocytes obtained from clofibrate-treated rat liver were used as an enzyme source, PCA 16-dependent production of H 2O 2 was increased by around 6-fold. The activity of peroxisomal β-oxidation for PCA 16 assayed by H 2O 2 generation was 3-fold higher than that for palmitic acid, whereas the activity of mitochondrial β-oxidation for PCA 16 assayed by ketone body production was much less than that for palmitic acid. A subcellular distribution study revealed that the distribution of the activities of β-oxidation and fatty acylCoA oxidase for PCA 16-CoA coincided with those of cyanide-insensitive palmitoyl-CoA-dependent β-oxidation and catalase, a marker enzyme of peroxisomes. The profile of the cofactor requirement for β-oxidation of PCA 16-CoA in isolated peroxisomes was similar to that for palmitoyl-CoA oxidation, and the reaction was not inhibited by KCN. The formation of CoA derivative prior to β-oxidation reaction was essential. HPLC analysis of metabolites after incubation of PCA 16-CoA with isolated peroxisomes demonstrated the production of four metabolites, two of which were identified as PCA 14 and PCA 12 by fast atomic bombardment-mass spectrometry. These results indicate that peroxisomal β-oxidation participates in the shortening of the alkyl-side chain of PCA 16 and plays an important role in the formation of antileukemic cytosine arabinoside from YNKO1.

Clofibrate induces carnitine acyltransferases in periportal and perivenous zones of rat liver and does not disturb the acinar zonation of gluconeogenesis

Clofibrate induces hypertrophy and hyperplasia and marked changes in the activities of various enzymes in rat liver. We examined the effects of treatment of rats with clofibrate on enzyme induction and on rates of metabolic flux in hepatocytes isolated from the periportal and perivenous zones of the liver. Clofibrate induced the activities of carnitine acetyltransferase (90-fold), carnitine palmitoyltransferase (3-fold) and NADP-linked malic enzyme (3-fold) to the same level in periportal as in perivenous hepatocytes, suggesting that these enzymes were induced uniformly throughout the liver acinus. Increased rates of palmitate metabolism and ketogenesis after clofibrate treatment were associated with: a more oxidised mitochondrial redox state; diminished responsiveness to glucagon and loss of periportal/perivenous zonation. Despite the marked liver enlargement and hyperplasia caused by clofibrate, the normal periportal/perivenous zonation of alanine aminotransferase and gluconeogenesis was preserved in livers of clofibrate-treated rats, indicating that clofibrate-induced hyperplasia does not disrupt the normal acinar zonation of these metabolic functions.

Effect of clofibrate treatment on lipid peroxidation in rat liver homogenate and subcellular fractions

1. 1. A study was made of the effect of hypolipidemic drug clofibrate on the level of lipid peroxidation in homogenates and subcellular fractions of rat liver. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde formation. 2. 2. It was shown that under the action of clofibrate the levels of Fe/ADP-ascorbate-, as well as t-butyl hydroperoxide (Bu′OOH)-induced lipid peroxidation were decreased in the whole and “post-nuclear” liver homogenates. Dilution of the homogenates prevented depressing effect of clofibrate on lipid peroxidation. 3. 3. Clofibrate significantly decreased the level of the Bu′OOH-dependent lipid peroxidation, but did not affect the activity of the Fe/ADP-ascorbate-induced reaction in rat liver mitochondria and microsomes. 4. 4. Peroxidative alteration of membrane lipids in vivo was evaluated by determining the extent of conjugated dienes formation (absorption at 233 nm). It was shown that clofibrate did not increase the level of ultraviolet absorption of lipids from rat liver subcellular fractions. 5. 5. The data obtained indicate that cytosol from the clofibrate treated rat liver contains a factor(s) which prevents lipid peroxidation in the mitochondria and microsomes.

Permeability of the peroxisomal membrane to cofactors of beta-oxidation. Evidence for the presence of a pore-forming protein.

Peroxisomes were purified from livers of clofibrate-treated rats. Permeability measurements on the isolated organelles revealed that peroxisomes are permeable to small solutes, including sucrose and the cofactors for fatty acid oxidation NAD+, CoA, ATP, and carnitine. The intraperoxisomal distribution volume was equal for all solutes. Peroxisomal solute uptake was rapid, not saturable and not visibly influenced by temperature. NAD+ and carnitine uptake in the solute accessible volume was not diminished by a variety of analogs and inhibitors. Subfractionation of peroxisomes and reconstitution of the subfractions into liposomes preloaded with solutes made the liposomes reconstituted with the integral membrane protein fraction, but not those reconstituted with the other subperoxisomal protein fractions, permeable to the same solutes that entered intact peroxisomes. Solute leakage from the preloaded liposomes was rapid and not visibly influenced by temperature. Leakage activity was destroyed by heat treatment of the integral membrane protein fraction and was not present in lipid extracts of the membrane. Separation of the integral membrane proteins on sucrose density gradients and reconstitution of the gradient fractions into liposomes indicated that the leakage activity was caused by a polypeptide of rather low molecular weight. The gradient distribution of leakage activity corresponded most closely to the presence of a 22- and a 28-kDa polypeptide. Our experiments indicate that the nonspecific permeability of the peroxisomal membrane to small solutes is based on the presence in the membrane of a nonselective pore-forming protein.

Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver.

We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate-cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S-translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.

A subcellular study of the clofibratei-nduced increase in coenzyme a concentration in rat liver

When clofibrate [ethyl 2-(4-chlorophenoxv)-2-methylpropionatel was administered subcutaneously to rats (600 mg kg per day for 3 days), the concentration of CoA and its acyl derivatives in the liver increased 2.5-fold. Forty-eight per cent of the total cellular CoA in the clofibrate-treated rat liver and 51 per cent in the control liver was found in the mitochondrial fraction. In order to study the intermediates of CoA synthesis, clofibrate-treated rats were injected with [ 3H]pantothenate intracardially and killed after 30 min, l or 2hr for determination of the incorporation of radioactivity into CoA and its precursors. The incorporation of pantothenate into CoA after 2 hr was 5.9-fold in the liver and 4.5-fold in the liver mitochondrial fraction as compared with the control values. Measurement of the pantothenate concentration and radioactivity in clofibrate-treated and control rat liver showed that the higher incorporation of [ 3H]pantothenate into CoA in clofibrate-treated rat liver cannot be the result of a higher specific radioactivity of pantothenate. It is therefore evident that clofibrate affects the CoA concentration by increasing the rate of synthesis, although the rate of CoA degradation is simultaneously decreased, as has been shown previously [9]. The present results indicate that clofibrate increases the total hepatic CoA concentration without affecting the intracellular compartmentation of CoA. The clofibrate-induced increase in the rate of CoA synthesis does not result in differences in the compartmentation of the intermediates of CoA synthesis.

Increased biosynthesis of CoA in the liver of rats treated with clofibrate.

The amounts of CoA in rat liver increased about three fold after treatment with clofibrate. About 10 of total CoA were long‐chain acyl‐CoA in normal rats, and slightly less in clofibrate‐treated animals. The wet weight and protein content of the liver increased by about 25%. From livers of clofibrate‐treated rats, mitochondrial and lysosomal fractions were obtained in increased amounts by conventional tissue fractionation techniques. The supernatants from such livers had rather high activities of marker enzymes for lysosomes and peroxisomes. No changes of the specific activities of the two first enzymes in the degradation pathway for CoA, i.e. acid phosphatase and nucleotide pyrophosphatase, were found in the liver homogenate from clofibrate‐treated rats. In the mitochondrial fraction from such livers, both (contaminating) activities were decreased. The degradation of CoA was increased in the supernatant, however, probably because of an increased leakage of acid phosphatase from lysosomes during the fractionation procedure. The synthesis of CoA from pantothenic acid was estimated by two different methods in vitro: by quantitation of CoA formed and by estimation of the incorporation of [14C]pantothenic acid into CoA. By both methods, CoA synthesis was found to be increased 3–4‐fold in livers from clofibrate‐treated rats. Dephospho‐CoA kinase was found to have a dual localization, with a minor pool in the mitochondrial fraction. This enzyme was only slightly increased in the clofibrate‐treated animals. The activity of pantothenate kinase was more than doubled after clofibrate treatment. This is thought to be the most important factor for the increase of CoA. CoA exerted feed‐back inhibition at this step in liver supernatant from normal and clofibrate‐treated rats.

Increased uptake of fatty acids by the isolated rat liver after raising the fatty acid binding protein concentration with clofibrate

Following the administration of clofibrate to rats, the concentration of Z protein or fatty acid binding protein in liver cytosol increases by 98 %. Ligandin concentration remains unchanged. Isolated perfused livers of clofibrate-treated rats take up free fatty acids from the perfusate at a significantly higher rate (+ 76 %) than controls. Lipid synthesis from radioactive fatty acids is not modified by clofibrate administration. The yield of plasma membranes obtained from liver homogenates as well as their lipid composition are similar in control and clofibrate treated livers. These results seem to exclude the possibility that the enhancement of FFA uptake could result from an indirect effect of the drug on FFA metabolism and/or plasma membrane surface and thus support the view that Z protein plays a role in intracellular fatty acid transport in the liver.

Metabolism of ethanol and sorbitol in clofibrate-treated rats

The rates of ethanol and sorbitol removal and the cytoplasmic and mitochondrial redox states of the liver were determined in female rats pretreated with clofibrate (ethyl- α- p-chlorophenoxyiso-butyrate) for 2–15 days. The drug significantly increased the rate of elimination of ethanol even within the first two days. A significant increase in liver mass took place within a week but cannot explain the initial increase in ethanol removal. Although the liver mass was increased by clofibrate, the rate of sorbitol removal was significantly decreased. A decrease in liver sorbitol dehydrogenase activity was also observed. The sum of the removal of ethanol and sorbitol, when they were simultaneously metabolized, was significantly decreased in clofibrate-treated rats as compared with control ones. Sorbitol inhibition of ethanol elimination was increased but ethanol inhibition of sorbitol elimination was abolished by clofibrate administration. Ethanol and sorbitol caused similar changes in cytoplasmic (lactate/pyruvate) and mitochondrial (β-OH-butyrate/acetoacetate) redox states of clofibrate-treated rat liver, as has been earlier observed in livers of control animals.