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Regulation of mouse liver microsomal esterases by clofibrate and sexual hormones
Abstract
Carboxylesterase activity was measured using six different substrates in microsomal preparations from female and ovariectomized female mice in order to evaluate the effects of female sex hormones on esterase expression. With three of the substrates (alpha-naphthyl acetate and esters 2 and 3), esterase activity was the same in both groups; however, with the others (p-nitrophenyl acetate and esters 1 and 4), there was a small increase in activity in ovariectomized females, compared with intact females. Castration of males followed by treatment with testosterone caused only transient increases in activity for four of the substrates (alpha-naphthyl acetate and esters 1, 2, and 3) and no change in activity for the other two (p-nitrophenyl acetate and ester 4). Treatment of male and female mice with the peroxisome proliferator clofibrate, with or without testosterone, resulted in increased hydrolysis of alpha-naphthyl acetate and p-nitrophenyl acetate, but little change for the other substrates. Clofibrate also induced alpha-naphthyl acetate and p-nitrophenyl acetate hydrolysis in castrated males, but clofibrate and testosterone administered together resulted in significant increases of activity with all substrates, which were greater than the additive effects of the two compounds administered separately. These results indicate that clofibrate causes significant alterations in the regulation of esterase activity, whereas sex hormones only cause small changes. However, it would seem that testosterone can synergize the effect of clofibrate in castrated males, resulting in higher levels of activity than with clofibrate alone. Finally, an overall increase in esterase activity might be due to a large increase in the activity of a few esterases or to a small increase in many esterases. Enzyme staining of native polyacrylamide gels reveals that the latter is true, with the majority of esterases present in mouse liver microsomes being induced to a small degree by clofibrate.
... One important regulator of chemical disposition in the liver is the peroxisome proliferator-activated receptor alpha (PPAR). In fact, the PPAR ligands, di-(2-ethylhexyl)phthalate and clofibrate, have been shown to induce hepatic Ces activity in mice and rats (Hosokawa et al., 1994;Parker et al., 1996). Subsequent analysis demonstrated that the PPAR agonist GW7647 can upregulate the mRNA levels of specific Ces subtypes, namely Ces 1d, 1e, 1f, 2c and 2e (Jones et al. 2013). ...
Hepatic carboxylesterases (Ces) catalyze the metabolism of drugs, environmental toxicants, and endogenous lipids and are known to be regulated by multiple nuclear receptors. Perfluorooctanoic acid (PFOA) is a synthetic fluorochemical that has been associated with dyslipidemia in exposed populations. In liver, PFOA can activate nuclear receptors such as PPARα and alter the metabolism and excretion of chemicals. Here, we sought to test the ability of PFOA to modulate Ces expression and activity in the presence and absence of the PPARα receptor. For this purpose, male C57BL/6 NCrl mice were administered PFOA (1 or 3 mg/kg, po, 7 days) and livers collected for assessment of Ces expression and activity. PFOA increased Ces1 and 2 protein and activity. Notably, PFOA increased Ces1d, 1e, 1f, 1 g, 2c, and 2e mRNAs between 1.5- and 2.5-fold, while it decreased Ces1c and 2b. Activation of PPARα by PFOA was confirmed by up-regulation of Cyp4a14 mRNA. In a separate study of PFOA-treated wild-type (WT) and PPARα-null mice, induction of Ces 1e and 1f mRNA and in turn, Ces1 protein, was PPARα-dependent. Interestingly, in PPARα-null mice, Ces1c, 1d, 1 g, 2a, 2b, and 2e mRNAs and Ces2 protein were up-regulated by PFOA which contributed to sustained up-regulation of Ces activity, although to a lower extent than observed in WT mice. Activation of the CAR and PXR receptors likely accounted for up-regulation of select Ces1 and 2 subtypes in PPARα-null mice. In conclusion, the environmental contaminant PFOA modulates the expression and function of hepatic Ces enzymes, in part through PPARα.
... Membrangebundene mikrosomale Esteraseaktivitäten sind neben zyto-solischen beschrieben (Helia et al., 1995;Hernandez et al., 1996;Parker et al., 1996). Es ist auch bekannt, daß Kompositmonomere durch Esteraseaktivitäten metabolisiert werden könnten Phillips et al., 1996;Bean und Williams, 1997). ...
... Fatty Acids and Fibrates/PPARs. Early studies in mice and rats demonstrated that certain drugs that enhanced hepatic peroxisome proliferation also caused an increase in microsomal carboxylesterase activity (Mentlein et al., 1986;Ashour et al., 1987;Hosokawa and Satoh, 1993;Parker et al., 1996). In particular, treating rodents with the PPARa ligands clofibrate and WY14,643, or perfluorinated fatty acids resulted in increased CES enzyme activity of five to six subclasses defined by substrate specificity. ...
Carboxylesterases (CES) are a well recognized, yet incompletely characterized family of proteins that catalyze neutral lipid hydrolysis. Some CES have well-defined roles in xenobiotic clearance, pharmacologic pro-drug activation, and narcotic detoxification. In addition, emerging evidence suggests other CES may have roles in lipid metabolism. Humans have six CES genes, while mice have 20 Ces genes grouped into five isoenzyme classes. Perhaps due to the high sequence similarity shared by the mouse Ces genes, the tissue-specific distribution of expression for these enzymes has not been fully addressed. Therefore, we performed studies to provide a comprehensive tissue distribution analysis of mouse Ces mRNAs. These data demonstrated that while the mouse Ces family 1 is highly expressed in liver and family 2 in intestine, many Ces genes have a wide and unique tissue distribution defined by relative mRNA levels. Furthermore, evaluating Ces gene expression in response to pharmacologic activation of lipid and xenobiotic-sensing nuclear hormone receptors (NHR) showed differential regulation. Finally, specific shifts in Ces gene expression were seen in peritoneal macrophages following LPS treatment and in a steatotic liver model induced by high-fat feeding, two model systems relevant to disease. Overall these data show that each mouse Ces gene has its own distinctive tissue expression pattern and suggest that some CES may have tissue-specific roles in lipid metabolism and xenobiotic clearance.
... suggested that carboxylesterase is regulated by testosterone in many animal species, including cats (Miyazaki et al. 2006), mice (Parker et al. 1996), and rats (Hosokawa et al. 1985), suggesting a possible sexual differentiation in response to treatment with oseltamivir. In summary, on the one hand, oseltamivir produced some burden on hepatic cells, reducing the activities of chemoprotective and (or) antioxidant enzymes and elevating hepatotoxicity biomarkers investigated, but on the other hand, oseltamivir was less likely to cause drug interactions or renal damage. ...
Oseltamivir is the most widely used antiviral drug for the treatment and prophylaxis of influenza. However, not much is known about its adverse effects. The potential side effects were investigated in male and female rats (140-170 g). Oseltamivir was administered at 2.2 mg·kg(-1)·day(-1) for 5 days. For both genders, treatment with oseltamivir resulted in significant reductions in the hepatic activities of glutathione reductase, glutathione peroxidase, and glutathione S-transferase. Also for both genders, oseltamivir produced modest reductions in the hepatic activities of UDP-glucuronosyltransferase, quinone oxidoreductase, thioredoxin reductase, CYP1A1/2, and CYP3A, as well as hepatic glutathione content. For both genders, neither the kidney functions nor protein profile was affected by oseltamivir. Oseltamivir also caused significant elevation in serum levels of both triacylglycerols and LDL-cholesterol and in the activity of γ-glutamyl transpeptidase, in both genders. For male animals only, oseltamivir treatment elevated the serum level of total cholesterol as well as the activity of serum alanine aminotransferase, and reduced the hepatic activities of superoxide dismutase and catalase. Oseltamivir caused oxidative stress and acute toxicity in the liver, and disrupted the cholesterol and lipid metabolism but was less likely to cause serious drug interactions. There was a sexual differentiation in these adverse effects, with adverse effects being more evident in male rats.
... Previously, 5 weeks of ex- posure to the PPARa agonist WY-14,643 reduced the expression of murine liver TGH, though differences in TGH expression were not observed at 1 and 3 weeks of exposure [132]. Studies utilizing different peroxisomal proliferator compounds, doses and length of administration observed increased TGH expression in rats [133,134] and mice [133,135]. It appears that the changes in TGH expression were observed only during long-term administration of a PPARa agonist and reflect a secondary response. ...
Recent scientific advances have revealed the identity of several enzymes involved in the synthesis, storage and catabolism of intracellular neutral lipid storage droplets. An enzyme that hydrolyzes stored triacylglycerol (TG), triacylglycerol hydrolase (TGH), was purified from porcine, human and murine liver microsomes. In rodents, TGH is highly expressed in liver as well as heart, kidney, small intestine and adipose tissues, while in humans TGH is mainly expressed in the liver, adipose and small intestine. TGH localizes to the endoplasmic reticulum and lipid droplets. The TGH genes are located within a cluster of carboxylesterase genes on human and mouse chromosomes 16 and 8, respectively. TGH hydrolyzes stored TG, and in the liver, the lipolytic products are made available for VLDL-TG synthesis. Inhibition of TGH activity also inhibits TG and apolipoprotein B secretion by primary hepatocytes. A role for TGH in basal TG lipolysis in adipocytes has been proposed. TGH expression and activity is both developmentally and hormonally regulated. A model for the function of TGH is presented and discussed with respect to tissue specific functions.
... It has been known that carboxylesterase isoforms are related to metabolism of drugs [16]. Clofibrate is known with inducer of alpha-naphthyl acetate that is a substrate of carboxylesterase which involved in the metabolism of xenobiotics and of natural substrates [12], and carboxylesterase has been known with activators of the peroxisome proliferator. It has been studied that microsomal carboxylesterases were increased by gemfibrozil [13]. ...
The recent DNA microarray technology enables us to understand a large number of gene expression profiling. The technology has potential possibility to comprehend mechanism of multiple genes were related to compounds which have toxicity in biological system. So, the toxicogenomics through this technology may be very powerful for understanding the effect of unknown toxic mechanisms in biological system. We have studied that the effect of compounds related to hepatotoxin in vivo system using DNA microarray and classified chemicals which have been well characterized. We have studied three compounds; 2 peroxisome proliferators: Clofibrate (ethyl-p-chlorophenoxyisobutyrate), gemfibrozil (5-2[2,5-dimethyl-phenoxy]2-2-dimethyl-pentanonic), and an antiepileptic drug: phenytoin (5,5-diphenylhydantoin). Male Sprague-Dawely VAF(+) albino rats of 5-6 weeks old were treated with each compound for 24 hr and 2 weeks. 4.8 K cDNA microarray in house has been used for gene expression profiling. We found that the clustering of gene expression had similarity like as the toxic phenotype of compounds.
Nonalcoholic fatty liver disease (NAFLD) is increasingly recognized as a global public health problem. Carboxylesterases (CESs), as potential influencing factors of NAFLD, are very important to improve clinical outcomes. This review aims to deeply understand the role of CESs in the progression of NAFLD and proposes that CESs can be used as potential targets for NAFLD treatment. We first introduced CESs and analyzed the relationship between CESs and hepatic lipid metabolism and inflammation. Then, we further reviewed the regulation of nuclear receptors on CESs, including PXR, CAR, PPARα, HNF4α and FXR, which may influence the progression of NAFLD. Finally, we evaluated the advantages and disadvantages of existing NAFLD animal models and summarized the application of CES-related animal models in NAFLD research. In general, this review provides an overview of the relationship between CESs and NAFLD and discusses the role and potential value of CESs in the treatment and prevention of NAFLD.
Bioprecursor by definition is a type of prodrug that is designed to tackle pharmacetical, pharmacokinetic or pharmacodynamics shortcomings of a drug that limits its clinical use. A retrometabolic approach is used to design bioprecursors which upon activation by either phase-I or phase-II metabolic enzymes result into an active metabolite. A bioprecursor differs from its active metabolite in many respects like physico-chemical, biological and toxicological aspects. Present review focuses on salient features of design and chemistry of bioprecursors and their active metabolites, activating metabolic enzymes/reactions, reported examples from literature and applications. We hope that this extensive compilation would open doors to bioprecursors and their active metabolites as very promising resources for the discovery of novel drugs possessing high efficacy with lower number and extent of adverse effects.
Accurate annotated assemblies of the mouse and human genomes enable a detailed comparison of the organization and evolution of the two genomes. We have completed several assemblies of both the mouse, with and without public data, and human genomes. Analysis of these assemblies suggests the mouse genome is about 10% smaller than the human genome primarily because of a difference in the content of repetitive DNA between the two genomes. More than 300,000 positions in these two genomes can be aligned with one another based on short segments of sequence similarity. These conserved segments significantly enhance the resolution of the resultant comparative maps and can be used to divide the genomes into regions of conserved-shared synteny. The genes found in such regions are highly conserved as is their relative order and orientation.Comparison of the human and mouse genome is expected to be key to deciphering the important biological information encoded in the mammalian genome. A prerequisite to comparing complex genomes such as those of mouse and human is the availability of annotated assemblies of both genomes that are comparable in quality and completeness. Since February 2001, we have assembled, annotated and delivered to our subscribers two versions of the human genome and two versions of the mouse genome. A third assembly of the human genome is being completed and will be delivered by fall of 2002. These annotated assemblies provide the starting materials for the genome-wide comparisons of the mouse and human reported here. We will begin with a description of the first Celera whole genome assembly of the mouse to provide a general basis of the quality and completeness of these data and then will report the results of a preliminary comparison between these two genomes.
Mammalian liver microsomal carboxylesterases comprise a family of isozymes, few of which have been purified or studied at the molecular level. These enzymes play an important role in the metabolism of drugs, lipids and other xenobiotics. The purpose of this study was to establish conditions for the selective solubilization of these microsomal enzymes.Solubilization of mouse liver microsomal carboxylesterase was examined with Triton X-100, Lubrol PX, octyl glucoside and 3-(3′-cholamidopropyl)-dimethylammonio-1-propane sulphonate (CHAPS). The solubilized esterase activities were assayed with p-nitrophenyl acetate (p-NpAc), -naphthyl acetate (-NA) and malathion. Triton X-100, Lubrol PX and CHAPS solubilized > 90% of the esterase activity acting on p-NpAc and malathion at 0·05–0·10% concentrations, whereas esterase activity acting on -NA was released at 0·3–1·0% concentrations. Octyl glucoside caused maximum solubilization of esterase activity acting on malathion at 0·3% and p-NpAc or -NA at > 1%.These detergents solubilized the membrane-bound esterases to varying degrees depending on the concentrations and the substrate used. Octyl glucoside and CHAPS are effective detergents for solubilization due to their high critical micellar concentrations, selectivity, maintenance of high esterase activities and ease of removal by dialysis.
Dimethacrylate derivatives are used as monomers to polymerize dental composite materials and for a great variety of other industrial resins. Occupational exposure is likely in various ways because of the many areas of methacrylate application. Here, the mutagenicity of the monomers, bisphenol A-diglycidyl dimethacrylate (Bis-GMA), urethane dimethacrylate (UDMA), triethylene glycol dimethacrylate (TEGDMA), Bisphenol A (BPA), glycidyl methacrylate (GMA), methyl methacrylate (MMA), and 2-hydroxyethyl methacrylate (HEMA) was studied in a bacterial (Ames test) and a mammalian gene mutation assay (V79/HPRT assay). Mutagenicity was determined in different Salmonella typhimurium strains (TA97a, TA98, TA100, TA102) and in V79 cells in the presence and in the absence of a metabolically active microsomal fraction from rat liver (S9). No mutagenic effects were observed with Bis-GMA and UDMA, methyl methacrylate, 2-hydroxyethyl methacrylate and bisphenol A. Glycidyl methacrylate (GMA) was mutagenic in a dose-dependent manner in three Salmonella tester strains. The number of mutants was increased by a factor of 2 to 3 with strains TA97a and TA102 in the absence of S9. Moreover, the numbers of mutants induced in S. typhimurium TA100 were about 8-fold higher than in solvent controls. GMA also induced an increase of mutants in V79 cells in the absence of S9. However, GMA was inactivated by microsomal enzymes. Triethylenglycol dimethacrylate (TEGDMA) was not mutagenic in any S. typhimurium. In contrast, the compound induced a dose-dependent rise in mutant frequencies in V79 cell cultures. It is concluded that TEGDMA acted through a clastogenic mechanism which is not detected by Ames tester strains.
The effects of clofibrate on the content and composition of liver and plasma lipids was studied in mice fed for 4 wk on diets enriched in n-6 or n-3 polyunsaturated fatty acids (PUFA) from sunflower oil (SO) or fish oil (FO), respectively; both oils were fed at 9% of the diet (dry weight basis). Only FO was hypolipidemic. Both oil regimes led to slightly increased concentrations of phospholipids (PL) and triacylglycerols (TG) in liver as compared with a standard chow diet containing 2% fat. Clofibrate promoted hypolipidemia only in animals fed SO. Its main effect was to enlarge the liver, such growth increasing the amounts of major glycerophospholipids while depleting the TG. SO and FO consumption changed the proportion of n-6 or n-3 PUFA in liver and plasma lipids in opposite ways. After clofibrate action, the PUFA of liver PL were preserved better than in the absence of oil supplementation. However, most of the drug-induced changes (e.g., increased 18:1n-9 and 20:3n-6, decreased 22:6/20:5 ratios) occurred irrespective of lipids being rich in n-6 or n-3 PUFA. The concentration of sphingomyelin (SM), a minor liver lipid that virtually lacks PUFA, increased with the dietary oils, decreased with clofibrate, and changed its fatty acid composition in both situations. Thus, oil-increased SM had more 22:0 and 24:0 than clofibrate-decreased SM, which was significantly richer in 22:1 and 24:1.
The nonspecific carboxylesterases (EC.3.1.1.1) are a large group of enzymes that play important roles in the metabolism of foreign xenobiotics and endogenous lipids, including activators of the peroxisome proliferator-activated receptor alpha, a nuclear receptor that is the central mediator of peroxisome proliferator (PP) effects in the rodent liver. A number of reports have demonstrated that PP exposure leads to alterations in levels of carboxylesterases in the liver. In this study, we determined by Western blot analysis whether exposure to diverse PP results in alteration of expression of two highly expressed microsomal carboxylesterases. Chronic exposure to the PP WY-14,643 (WY) and gemfibrozil (GEM), but not di-n-butyl phthalate (DBP), led to decreases in ES-4 in male rat livers. ES-4 was increased in female rat livers treated with GEM. WY exposure led to decreases in ES-10 in male and female rat livers. ES-10 was increased in female rats treated with DBP. Compared with other end points that are altered within days after PP exposure, the downregulation of ES-4 and ES-10 by WY was considerably slower, occurring between 1 and 5 weeks of exposure. Decreased expression of ES-4 was observed at doses of WY or GEM as low as 10 or 8000 ppm, respectively, whereas decreased expression of ES-10 was more resistant to changes by any PP occurring only with WY at doses as low as 50 ppm. After chronic exposure to WY or diethylhexyl phthalate in wild-type mice, kidney, but not liver, expression of ES-4 and ES-10 was downregulated. These decreases in kidney ES expression were not observed in PPARalpha-null mice lacking a functional PPARalpha gene, demonstrating the importance of this transcription factor in these changes. These studies demonstrate that ES protein expression is under complex control by PP that is sex- and compound-dependent. These results lend support to the hypothesis that PP exposure leads to a reprogramming of expression of enzymes important in the metabolism of PPARalpha activators.
Triacylglycerol hydrolase (TGH) is an enzyme that catalyzes the lipolysis of intracellular stored triacylglycerol (TG). Peroxisomal proliferator-activated receptors (PPAR) regulate a multitude of genes involved in lipid homeostasis. Polyunsaturated fatty acids (PUFA) are PPAR ligands and fatty acids are produced via TGH activity, so we studied whether dietary fats and PPAR agonists could regulate TGH expression. In 3T3-L1 adipocytes, TGH expression was increased 10-fold upon differentiation, compared to pre-adipocytes. 3T3-L1 cells incubated with a PPARgamma agonist during the differentiation process resulted in a 5-fold increase in TGH expression compared to control cells. Evidence for direct regulation of TGH expression by PPARgamma could not be demonstrated as TGH expression was not affected by a 24-h incubation of mature 3T3-L1 adipocytes with the PPARgamma agonist. Feeding mice diets enriched in fatty acids for 3 weeks did not affect hepatic TGH expression, though a 3-week diet enriched in fatty acids and cholesterol increased hepatic TGH expression 2-fold. Two weeks of clofibrate feeding did not significantly affect hepatic TGH expression or microsomal lipolytic activities in wild-type or PPARalpha-null mice, indicating that PPARalpha does not regulate hepatic TGH expression. Therefore, TGH expression does not appear to be directly regulated by PPARs or fatty acids in the liver or adipocytes.
Peroxisome proliferators are postulated to elicit predictable pleiotropic responses in the liver by activating a peroxisome proliferator-activated receptor (PPAR). PPARs from mouse liver (mPPAR), rat liver (rPPAR), and Xenopus liver (xPPAR gamma) have been cloned recently. We now report the cloning of a new member from mouse liver which we designate mPPAR gamma. mPPAR gamma cDNA contained an open reading frame encoding a 475-amino acid protein exhibiting 75% amino acid similarity to xPPAR gamma, while it showed only 55% identity with mPPAR. The ligand-binding and DNA-binding domains are best conserved between mPPAR gamma, mPPAR, and xPPAR gamma. Like rPPAR, mPPAR gamma is able to impart peroxisome proliferator responsiveness to the promoter of peroxisomal bifunctional gene, which encodes the second enzyme of the peroxisomal fatty acid beta-oxidation system. Northern blot analysis revealed high expression of mPPAR gamma gene in mouse liver, kidney, and heart and low expression in the lung, testis, brain, skeletal muscle, and spleen. In mice treated with ciprofibrate, a peroxisome proliferator, a 2-fold increase in mPPAR gamma mRNA was observed in the liver and kidney. The presence of two PPARs in the mouse liver suggests the possibility of multiple signaling pathways for the peroxisome proliferator-induced pleiotropic responses.
Apparent Induction of Microsomal Carboxylesterase Activities in Tissues of Clofibrate-Fed
and Rats. ASHOUR,M.-B.A., MOODY, D. E., AND HAMMOCK,B. D. (1987). Toxicol. Appl. Pharmacol. 89, 361-369. Treatment with 0.5% (w/w) dietary clofibrate, a peroxisome
proliterator, for 14 days induced microsomal carboxylesterase activities for five substrates including malathion, clofibrate, diethylsuccinate, diethylphthalate, and pnitrophenylacetate in liver and kidney of male Swiss-Webster mice and Sprague-Dawley rats. The induction was substrate, tissue, and species dependent. The carboxylesterase activity was induced in mouse from 1.2- to 2.2-fold (liver) and from 1 . 1 - to 1.7-fold (kidney) depending upon substrate used. Analogous values from rat ranged from 1 .O- to 1.4-fold (liver) and from 1 . l- to 1 .&fold (kidney). Enzyme activities were either decreased or not affected in testes of treated mice and rats. Substituted trifluoroketones (“transition-state” inhibitors of carboxylesterase) were found to be very potent inhibitors of clofibrate-metabolizing carboxylesterase(s) and to be potentially useful in distinguishing among isozymes. The inhibition data suggested that changes in carboxylesterase activity following clofibrate treatment were. both qualitative and quantitative. 8 1987 Academic Mice Press. Inc.
The liver cells of intact male rats given ethyl-α-p-chlorophenoxyisobutyrate (CPIB) characteristically show a marked increase in microbodies and in catalase activity, while those of intact female rats do not. In castrated males given estradiol benzoate and CPIB the increase in catalase activity and microbody proliferation is abolished, while in castrated females given testosterone propionate and CPIB the livers show a marked increase in microbodies and in catalase activity. No sex difference in microbody and catalase response is apparent in fetal and neonatal rats. Both sexes show a sharp rise in catalase activity on the day of birth, with a rapid decline at 5 days after birth. Thyroidectomy abolishes the hypolipidemic effect of CPIB in rats, but microbody proliferation and increase in catalase activity persists in thyroidectomized male rats, indicating that microbody proliferation can be independent of hypolipidemia. Adrenalectomy does not alter appreciably the microbody-catalase response to CPIB. These experiments demonstrate that (1) in adult rats, hepatic microbody proliferation is dependent to a significant degree upon male sex hormone but is largely independent of thyroid or adrenal gland hormones; (2) hepatic microbody proliferation is independent of the hypolipidemic effect of CPIB; (3) displacement of thyroxine from serum protein may not be sufficient cause for stimulation of microbody formation.
Twenty-two soluble esterases have been identified inD. melanogaster by combining the techniques of native polyacrylamide gel electrophoresis and isoelectric focusing. The sensitivity of each isozyme to three types of inhibitors (organophosphates, eserine sulfate, and sulfydryl reagents) identified 10 as carboxylesterases, 6 as cholinesterases, and 3 as acetylesterases. Three isozymes could not be classified and no arylesterases were identified. The carboxyl- and cholinesterases could each be further divided into two subclasses on the basis of inhibition by organophosphates and sulfhydryl reagents, respectively. Cholineand acetylesterases have characteristic substrate preferences but both subclasses of carboxylesterases are heterogeneous in substrate utilization. Subclass 2 carboxylesterases exhibit diverse temporal expression patterns, with subclass 1 carboxylesterases generally found in larvae and subclass 1 cholinesterases and acetylesterases more characteristic of pupae and adults. Tissues showing the greatest number of isozymes are larval body wall (eight) and digestive tract (six in larvae, six in adults). Carboxylesterases are distributed across a wide range of tissues, but subclass 1 cholinesterases are generally associated with neural or neurosecretory tissues and subclass 2 cholinesterases with digestive tissues.
Peroxisome proliferators are a diverse group of chemicals, including several hypolipidaemic drugs, that activate a nuclear hormone receptor termed the peroxisome proliferator activated receptor (PPAR). The peroxisomal enzyme acyl CoA oxidase (ACO) is the most widely used marker of peroxisome proliferator action. We have examined the 5' flanking region of the rat ACO gene for sequences that mediate the transcriptional effect of peroxisome proliferators and have identified an element located 570 bp upstream of the ACO gene that confers responsiveness to the hypolipidaemic peroxisome proliferator Wy-14,643. This peroxisome proliferator response element (PPRE) contains a direct repeat of the sequence motifs TGACCT and TGTCCT and binds PPAR. These data therefore indicate an important role of PPAR in mediating the action of peroxisome proliferators including the induction of ACO.
Peroxisome proliferators are hepatocarcinogens in rats and mice. Chronic administration of these compounds results in the development of altered areas and neoplastic nodules followed by hepatocellular carcinomas. All three types of hepatic lesions do not express gamma-glutamyltranspeptidase, glutathione 8-transferase-P, and alpha-fetoprotein and are resistant to iron accumulation after overload. The mechanism by which nongenotoxic peroxisome proliferators induce hepatic tumors is not well understood. It has been proposed that with continuous administration of peroxisome proliferators, liver cells are subjected to persistent oxidative stress resulting from marked proliferation of peroxisomes and a differential increase in the levels of H2O2 producing (20- to 30-fold) and degrading (2-fold) enzymes. Free oxygen radicals lead to DNA damage (both directly and through lipid peroxidation) and thus may cause initiation and promotion of the carcinogenic process.
The induction of liver cytochrome P450 4A-catalyzed fatty acid omega-hydroxylase activity by clofibrate and other peroxisome proliferators has been proposed to be causally linked to the ensuing proliferation of peroxisomes in rat liver. Since female rats are less responsive than males to peroxisome proliferation induced by clofibrate, the influence of gender and hormonal status on the basal and clofibrate-inducible expression of the 4A P450s was examined. Northern blot analysis using gene-specific oligonucleotide probes revealed that in the liver, P450 4A1 and 4A3 mRNAs are induced to a much greater extent in male as compared to female rats following clofibrate treatment, whereas P450 4A2 mRNA is altogether absent from female rat liver. Male-specific expression of P450 4A2 mRNA was also observed in kidney. Western blot analysis indicated that a similar sex dependence characterizes both the basal expression and the clofibrate inducibility of the corresponding P450 4A proteins. This suggests that the lower responsiveness of female rats to clofibrate-induced peroxisome proliferation may reflect the lower inducibility of the P450 4A fatty acid hydroxylase enzymes in this sex. Investigation of the contribution of pituitary-dependent hormones to the male-specific expression of 4A2 revealed that this P450 mRNA is fully suppressed in liver following exposure to the continuous plasma growth hormone profile that characterizes adult female rats; in this and other regards liver P450 4A2 is regulated in a manner that is similar, but not identical to, P450 3A2, a male-specific testosterone 6 beta-hydroxylase. In contrast, kidney 4A2 expression, although also male-specific, was not suppressed by continuous growth hormone treatment, but was regulated by pathways that, in part, involve testosterone as a positive regulator. The male-specific expression of liver and kidney P450 4A2 is thus under the control of distinct pituitary-dependent hormones acting in a tissue-specific manner.
The peroxisome proliferator-activated receptor (PPAR) is a member of the steroid hormone receptor superfamily and is activated by a variety of fibrate hypolipidaemic drugs and non-genotoxic rodent hepatocarcinogens that are collectively termed peroxisome proliferators. A key marker of peroxisome proliferator action is the peroxisomal enzyme acyl CoA oxidase, which is elevated about ten fold in the livers of treated rodents. Additional peroxisome proliferator responsive genes include other peroxisomal beta-oxidation enzymes and members of the cytochrome P450 IVA family. A peroxisome proliferator response element (PPRE), consisting of an almost perfect direct repeat of the sequence TGACCT spaced by a single base pair, has been identified in the upstream regulatory sequences of each of these genes. The retinoid X receptor (RXR) forms a heterodimer with PPAR and binds to the PPRE. Furthermore, the RXR ligand, 9-cis retinoic acid, enhances PPAR action. Retinoids may therefore modulate the action of peroxisome proliferators and PPAR may interfere with retinoid action, perhaps providing one mechanism to explain the toxicity of peroxisome proliferators. Interestingly, a variety of fatty acids can activate PPAR supporting the suggestion that fatty acids, or their acyl CoA derivatives, may be the natural ligands of PPAR and that the physiological role of PPAR is to regulate fatty acid homeostasis. Taken together, the discovery of PPAR has opened up new opportunities in understanding how lipid homeostasis is regulated, how the fibrate hypolipidaemic drugs may act and should lead to improvements in the assessment of human risk from peroxisome proliferators based upon a better understanding of their mechanism of action.
Peroxisome proliferators are postulated to elicit predictable pleiotropic responses in the liver by activating a peroxisome proliferator-activated receptor (PPAR). PPARs from mouse liver (mPPAR), rat liver (rPPAR), and Xenopus liver (xPPAR gamma) have been cloned recently. We now report the cloning of a new member from mouse liver which we designate mPPAR gamma. mPPAR gamma cDNA contained an open reading frame encoding a 475-amino acid protein exhibiting 75% amino acid similarity to xPPAR gamma, while it showed only 55% identity with mPPAR. The ligand-binding and DNA-binding domains are best conserved between mPPAR gamma, mPPAR, and xPPAR gamma. Like rPPAR, mPPAR gamma is able to impart peroxisome proliferator responsiveness to the promoter of peroxisomal bifunctional gene, which encodes the second enzyme of the peroxisomal fatty acid beta-oxidation system. Northern blot analysis revealed high expression of mPPAR gamma gene in mouse liver, kidney, and heart and low expression in the lung, testis, brain, skeletal muscle, and spleen. In mice treated with ciprofibrate, a peroxisome proliferator, a 2-fold increase in mPPAR gamma mRNA was observed in the liver and kidney. The presence of two PPARs in the mouse liver suggests the possibility of multiple signaling pathways for the peroxisome proliferator-induced pleiotropic responses.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Plasticizers (e.g. di-(2-ethylhexyl)-pthalate (DEHP)) in common use in PVC plastics and hypolipidemic drugs (e.g. clofibrate) are two types of compound is to which we may be frequently exposed. Yet these are among a group of diverse compounds which cause hepatic peroxisome proliferation, a condition which can lead to hepatic cancer by non-mutagenic means. Janardan Reddy and Sambasiva Rao discuss the possible mechanisms of induction of peroxisome proliferator pleiotropic responses leading to the development of liver tumors, focusing in particular on the hypothesis that the mechanism involves a single biological receptor, or a set of receptors.
NMRI mice were treated with testosterone propionate (TSP) and PCB. The effect on nonspecific esterase activity and the relative activities of the various molecular forms of nonspecific esterases were noted. The activity of most molecular forms is sex specific in serum, liver and kidney. Administration of TSP affects the sex specificity, especially in serum. The kidney is most sensitive to TSP. There is a general difference between liver and kidney in the response to TSP. Administration of PCB causes changes in activity and distribution of different molecular forms. There seems to be no connection between TSP and PCB in their effects on nonspecific esterases.
The main polymorphic system of esterase isoenzymes in adults of the G3 laboratory strain ofAnopheles gambiae consists of two to five major bands of activity per individual. The bands are designated 5S, 5F, 13, 14, and 15. In genetic
crosses, the genes which coded for the bands assorted as three codominant alleles, Est A, Est B, and Est C, at a single autosomal
locus. Homozygotes for the Est C allele were significantly underrepresented among backcross progeny. The developmental pattern
of esterase expression was examined. Esterase gene expression in embryos was first detectable between 2 and 12 hr after oviposition.
The initiation or termination of expression of some of the bands corresponded to boundaries between developmental stages.
Most of the esterase fractions were not specifically localized within the tissues tested, with the exception of a series of
bands which were restricted largely to adult male testes.
Treatment of rats with dehydroepiandrosterone (300 mg/kg body weight, peros, 14 days) caused a remarkable increase in the number of peroxisomes and peroxisomal ß-oxidation activity in the liver. The activities of carnitine acetyltransferase, microsomal laurate 12-hydroxylation, cytosolic palmitoyl-CoA hydrolase, malic enzyme and some other enzymes were also increased. The increases in these enzyme activities were all greater in male rats than in female rats. Immunoblot analysis revealed remarkable induction of acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme in the liver and to a smaller extent in the kidney, whereas no significant induction of these enzymes was found in the heart. The increase in the hepatic peroxisomal ß-oxidation activity reached a maximal level at day 5 of the treatment of dehydroepiandrosterone and the increased activity rapidly returned to the normal level on discontinuation of the treatment. The increase in the activity was also dose-dependent, which was saturable at a dose of more than 200 mg/kg body weight. All these features in enzyme induction caused by dehydroepiandrosterone correlate well with those observed in the treatment of clofibric acid, a peroxisome proliferator. Co-treatment of dehydroepiandrosterone and clofibric acid showed no synergism in the enhancement of peroxisomal ß-oxidation activity, suggesting the involvement of a common process in the mechanism by which these compounds induce the enzymes. These results indicate that dehydroepiandrosterone is a typical peroxisome proliferator. Since dehydroepiandrosterone is a naturally occurring C19 steroid in mammals, the structure of which is novel compared with those of peroxisome proliferators known so far, this compound could provide particular information in the understanding of the mechanisms underlying the induction of peroxisome proliferation.
A photometric method for determining acetylcholinesterase activity of tissue extracts, homogenates, cell suspensions, etc., has been described. The enzyme activity is measured by following the increase of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion. It is based on coupling of these reactions: The latter reaction is rapid and the assay is sensitive (i.e. a 10 μ1 sample of blood is adequate). The use of a recorder has been most helpful, but is not essential. The method has been used to study the enzyme in human erythrocytes and homogenates of rat brain, kidney, lungs, liver and muscle tissue. Kinetic constants determined by this system for erythrocyte eholinesterase are presented. The data obtained with acetylthiocholine as substrate are similar to those with acetylcholine.
A series of substituted trifluoroketones were tested as inhibitors of mammalian liver microsomal carboxylesterase(s) hydrolyzing a variety of substrates including malathion, diethylsuccinate (DES) and p-nitrophenyl acetate (p-NpAc). The trifluoroketones used were very potent “transition state” inhibitors of crude mouse and human liver microsomal carboxylesterases as well as commercial porcine liver carboxylesterase (Sigma EC 3.1.1.1 Type I). These enzymes were found to differ in their sensitivity to the inhibitors employed, and some compounds caused dramatic activation of the hydrolysis of DES. In some but not all cases, a thioether beta to the carbonyl increased the inhibitory potency of the compound. Structure-activity relationships also were evaluated among aliphatic versus substituted and unsubstituted aromatic trifluoroketones. Kinetic parameters [i.e. ] for the mouse liver microsomes and the porcine carboxylesterase hydrolyzing DES were determined. Apparent high-and low-affinity forms were observed with each preparation. 3-Nonylthio-1,1,1-trifluoropropan-2-one was synthesized by the reaction of the corresponding thiol with 3-bromo-1,1,1-trifluoroacetone, and apparent synergism was observed when it was coadministered i.p. with malathion to mice.
Induction of peroxisomal β-oxidation by clofibrate under altered hormonal states was investigated in female rat liver. Treatment of rats with clofibric acid (CPIB) caused a significant increase in hepatic peroxisomal β-oxidation, with female rats being less responsive than males (4.2- vs 12.2-fold increase). However, testosterone treatment following ovariectomy of female rats resulted in an enhanced response to CPIB, giving an induction (11.7-fold) comparable to that seen in male rats. Hypophysectomy of female rats also enhanced the induction (8.2-fold compared with 5.1-fold), suggesting a suppressive effect of a pituitary-dependent factor on CPIB induction of peroxisomal β-oxidation. Continuous infusion of growth hormone to the hypophysectomized female rats suppressed the enhanced induction nearly to the initial level (6.1-fold). The stimulatory effects of testosterone and hypophysectomy on the enzyme induction were additive. These findings suggest the involvement of growth hormone, as well as male sex hormone, in regulating the responsiveness to CPIB induction of peroxisomal β-oxidation in rat liver.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
With regard to the acyl moiety, carboxylesterases preferentially split esters of short-chain carboxylic esters. With most carboxylesterases the maximum of the reaction rate is found at an acyl chain length of C3 to C624,77,170,303 (see Section 5.4). However, there are some reports of carboxylesterases acting on medium- and long-chain fatty acid esters as well.143,189,252,253
Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. Testing of compounds related to lipid metabolism or peroxisomal proliferation revealed that 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activate the receptor chimera. In addition, saturated fatty acids induce the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. In conclusion, the present results indicate that fatty acids can regulate gene expression mediated by a member of the steroid nuclear receptor superfamily.
We have identified a novel member of the steroid hormone receptor superfamily by cDNA cloning from a human osteosarcoma SAOS-2/B10 cell library. Sequence analysis predicts a protein of 441 amino acids, which includes the conserved amino acid residues characteristic of the DNA- and ligand-binding domains of nuclear receptors. Amino acid sequence alignment and transcriptional activation experiments revealed that the new protein is closely related to the mouse peroxisome proliferator activated receptor. The overall homology is 62%, and the highest similarity is seen in the DNA- and ligand-binding domains, 86% and 71%, respectively. Northern blot analysis showed that in mature rats, the receptor is highly expressed in heart, kidney, and lung as a transcript of approximately 3500 nucleotides. In human cells, the size of the mRNA is approximately 4000 nucleotides. Transcription assays using hybrid receptors consisting of the ligand-binding domain of the new protein and the DNA-binding domain of the glucocorticoid receptor showed weak stimulation by the peroxisome proliferator activator WY14643, suggesting a relationship to that receptor. Similar stimulation was observed with arachidonic and oleic acid (100-250 microM).
We report here the identification of thyroid hormone response elements (TREs) that consist of a direct repeat, not a palindrome, of the half-sites. Unlike palindromic TREs, direct repeat TREs do not confer a retinoic acid response. The tandem TRE can be converted into a retinoic acid response element by increasing the spacing between the half-sites by 1 nucleotide, and the resulting retinoic acid response element is no longer a TRE. Decreasing the half-site spacing by 1 nucleotide converts the TRE to a vitamin D3 response element, while eliminating response to T3. These results correlate well with DNA-binding affinities of the thyroid hormone, retinoic acid, and vitamin D3 receptors. This study points to the general importance of tandem repeat hormone response elements and suggests a simple physiologic code exists in which half-site spacing plays a critical role in achieving selective hormonal response.
We have cloned a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The receptor homologue is activated by a diverse class of rodent hepatocarcinogens that causes proliferation of peroxisomes. Identification of a peroxisome proliferator-activated receptor should help elucidate the mechanism of the hypolipidaemic effect of these hepatocarcinogens and aid evaluation of their potential carcinogenic risk to man.
Inductions by perfluoro-octanoic acid (PFOA) of hepatomegaly, peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase were compared in liver between male and female rats. Marked inductions of these four parameters were seen concurrently in liver of male rats, whereas the inductions in liver of female rats were far less pronounced. The sex-related difference in the response of rat liver to PFOA was much more marked than that seen with p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). Hormonal manipulations revealed that this sex-related difference in the inductions is strongly dependent on sex hormones, namely that testosterone is necessary for the inductions, whereas oestradiol prevented the inductions by PFOA.
This review briefly discusses the response, in biochemical and morphological terms, of hepatocytes to peroxisome proliferating chemicals and then focuses on possible biochemical mechanism(s) whereby these chemicals may produce peroxisome proliferation.
The levels of hepatic carboxylesterases, including palmitoyl-CoA hydrolase and decanoyl-D,L-carnitine hydrolase, were studied in total homogenates and subcellular fractions prepared from the livers of male rats fed diets containing 0.3% clofibrate. The microsomal carboxylesterase as well as the fatty acyl-thioesterase are differently induced by clofibrate feeding. The specific activities of acetanilide carboxylesterase and decanoyl-D,L-carnitine hydrolase increased more than 3-fold in the microsomal fraction, compared to pellet-fed control animals. The microsomal activities of palmitoyl-CoA hydrolase and propanidid hydrolase were decreased by about 20 to 40% in clofibrate-treated rats. The specific clofibrate hydrolase activity remained unchanged after clofibrate administration, indicating that this microsomal carboxylesterase is not induced by its own substrate. The data suggest a different distribution of the differing carboxylesterase along the endoplasmic reticulum.
Hepatic microsomal carboxylesterase (EC 3.1.1.1) from rat liver microsomes showed a different capacity for the hydrolysis of various substrates. In hypophysectomized male rats, hepatic carboxylesterase activities showed substrate-dependent changes, i.e., an decrease in malathion and p-nitrophenylacetate hydrolases and increase in isocarboxazid hydrolase. When human growth hormone is administered to hypophysectomized male rats, carboxylesterase activities were decreased. Conversely, subcutaneous injection of ovine prolactin to hypophysectomized rats, carboxylesterase activities showed substrate-dependent changes. On the other hand, in hypophysectomized male rats, the amount of carboxylesterase isozymes showed different changes, i.e., an increase in the amount of RL2 contents and decrease in RL1 contents, and the amount of RH1 was not changed. These results suggested that hepatic microsomal carboxylesterases are regulated, at least in part, by some pituitary hormones, and that these hormones have selective effects on the different isozymes of carboxylesterase.
Three isozymes of carboxylesterase were purified from rat liver microsomes by using Sephadex G-150 gel filtration, DE-52 ion exchange, and chromatofocusing column chromatographies. These isozymes each showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were immunologically different from each other as determined by immunochemical blotting analysis and immunochemical inhibition of catalytic activity. The three isozymes were named RH1 (molecular weight 174,000, trimer, pl 6.0), RL1 (molecular weight 61,000, monomer, pl 6.5), and RL2 (molecular weight 61,000, monomer, pl 5.5). RL1 has the highest specific activities toward p-nitrophenylacetate and malathion. Acetanilide is a rather specific substrate for RL2, whereas RH1 has the highest specific activity for butanilicaine. RL1 has the highest specific activity for the hydrolysis of long-chain acyl-CoA. To investigate the hormonal regulation of carboxylesterase activities, we have quantitated RL1, RL2, and RH1 in liver microsomes from male and female rats using a radial immunodiffusion assay. The amount of RL1 in male rats was decreased by castration but recovered to almost the level in sham-operated rat liver microsomes after treatment of the castrated rats with testosterone. Conversely, in ovariectomized female rats, the amount of RL1 was increased as compared to that in sham-operated rats, and treatment of the ovariectomized rats with estradiol tended to decrease the quantity of RL1. In all cases of sex hormone treatment, the amount of RH1 remains unclear at present. However, the amount of RL2 may be, at least in part, regulated by estrogens. On the other hand, phenobarbital treatment of male and female rats caused a significant increase in the amounts of RH1 and RL2, whereas RL1 was not affected. It was concluded that the three isozymes differ considerably from each other in response to hormone treatment, inducibility, substrate specificity, and immunological properties.
In this critical review, I would like to provide a brief outline of the morphology, biochemical composition, distribution, and functions of peroxisomes. The induction of peroxisome proliferation and peroxisome-associated enzymes in the rodent liver by two classes of chemicals (hypolipidemic drugs and the industrial plasticizers) will be considered. The role of peroxisomes in lipid metabolism will be discussed. Carcinogenicity studies in rats and mice with these peroxisome proliferators will be evaluated critically. Careful consideration will be given to the hypothesis that "potent hepatic peroxisome proliferators as a class are carcinogenic." The possible mechanism(s) by which peroxisome proliferators induce liver tumors will be outlined. Particular attention will be paid to the possible role of peroxisome proliferation-mediated radical toxicity and generation of endogenous initiators of carcinogenesis.
Hepatic microsomal carboxylesterase (E.C. 3.1.1.1) from rat liver microsomes showed a different capacity for the hydrolysis of various substrates. In castrated male rats, the enzyme activities towards p-nitrophenylacetate and malathion were decreased. When testosterone propionate was administered to castrated male rats, the activities of p-nitrophenylacetate and malathion hydrolases were reversely increased. However, in ovariectomized female rats, the carboxylesterase activities showed substrate-dependent changes, i.e., increase in p-nitrophenylacetate and malathion hydrolases and decrease in acetanilide and isocarboxazid hydrolases. When estradiol benzoate was administered to ovariectomized female rats, the activities of p-nitrophenyl-acetate and malathion hydrolases were decreased; and acetanilide and isocarboxazid hydrolases were increased. These results suggest that hepatic microsomal carboxylesterases may be, at least in part, regulated by gonadal hormones which exert different effects on the several isozymes of carboxylesterases.
Rat liver microsomes contain many serine hydrolases, which can be demonstrated in electropherograms with carboxylesterase stain and with an active-site-directed radioactive organophosphate. Five of the most prominent of these enzymes plus dipeptidyl aminopeptidase IV, a microsomal serine hydrolase without activity against simple esters, have been highly purified with a simultaneous procedure after solubilization with saponin. The five carboxylesterases belong to at least three groups of chemically different proteins. Terminal amino acids, amino acid composition, and substrate specificity are different, while the subunit molecular weight of all esterases is very similar (about 60,000). All purified carboxylesterases have monooleylglycerol-cleaving capacity. The subunit weight (84,000) and the N-terminal amino acid (serine) of the peptidase differ from those of all isolated carboxylesterases. The data are correlated to other reports on individual serine hydrolases from rat liver.
Seventeen genes controlling the expression of carboxylic ester hydrolases, commonly known as esterases, have been identified in the mouse Mus musculus. Seven esterase loci are found on chromosome 8, where two clusters of esterase loci occur. It seems probable that the genes within these clusters have arisen from a common ancestral gene by tandem duplication. Close linkage of esterase genes is also found in the rat, rabbit, and prairie vole. Some mouse esterases appear to be homologous with certain human esterases. The function of these nonspecific enzymes is still unknown.
The distribution of molecular forms of esterases in serum, liver and kidney from NMRI, C57BL/6 and A/J mice and the effect of testosterone propionate on the zymograms were investigated. It was found that the different tissues are specific in the pattern of esterases and that, especially in serum and kidney, the distribution of esterases is highly sex specific. Sex hormones are responsible for this. Administration of testosterone has an effect on the amounts of esterases in both sexes, primarily reducing the normal sex differences.
Soluble epoxide hydrolase (sEH) activity was measured in the liver and kidneys of male, female, and castrated male mice in order to evaluate sex- and tissue-specific differences in enzyme expression. sEH activity was found to be higher in liver than in kidneys. Activity increased with age in the liver of females, males and castrated males, but only in males did activity in the kidneys increase. There was greater activity in both the liver and kidneys of adult males than females. This sexual dimorphism was more pronounced in the kidneys (283% higher) than in the liver (55% higher). Castration of males led to a decrease in activity in both organs, but it had a greater effect on renal activity (67% decrease) than on hepatic activity (27% decrease). Treatment of castrated mice with testosterone led to an increase in sEH activity of 400% in kidneys and 49% in liver compared with surgical controls. These results suggest differential regulation of sEH by testosterone in kidneys and liver. Ovariectomized female mice had renal and hepatic activities approximately 30% greater than control females. Feeding mice with the hypolipidemic drug clofibrate produced stronger induction of sEH in liver than in kidneys. Testosterone treatment, however, caused greater induction in kidneys than in liver of females and castrated males and had no effect in either kidneys or liver in males. When given together, the effects of these two compounds appeared to be additive in both liver and kidneys. Results from western blot showed that the increase in sEH enzyme activity in kidneys is correlated with an increase in sEH protein. These results suggest that clofibrate and testosterone independently regulate sEH activity in vivo, and that kidneys and liver respond differently to clofibrate and testosterone.
Hepatic peroxisome proliferation is induced by a number of agents, including clofibrate. Sustained proliferation of peroxisomes is associated with the development of hepatocellular carcinoma. In the present study, we have investigated the role of testosterone in peroxisome proliferation induced by clofibrate. Three groups of male rats (intact, castrated, and castrated replaced with testosterone) were studied. Proliferation of peroxisomes was induced by feeding clofibrate (0.25%, 0.50%, and 1.0% of diet) for 2 weeks. Peroxisome proliferation was monitored by measuring total peroxisomal beta-oxidation activity. In intact rats, the peroxisomal beta-oxidation activity (nmol/min/mg protein) increased in a dose-dependent manner and was 7.2 +/- 0.4, 52.6 +/- 7.5, 63.2 +/- 3.7, and 92.4 +/- 4.0 at clofibrate doses of 0%, 0.25%, 0.50%, and 1.0%, respectively. In contrast, in castrated rats, the total peroxisomal beta-oxidation activity was significantly (P < .01) lower at clofibrate levels of 0.25% and 0.50% (25.8 +/- 2.7 and 42.5 +/- 2.2, respectively), but not at the clofibrate level of 1.0% (85.0 +/- 6.3). Testosterone replacement of castrated rats restored the peroxisomal beta-oxidation activity. To determine whether the above results were related to the metabolism of clofibrate in the absence or presence of testosterone, we measured serum clofibrate levels. These levels were 50% lower in castrated rats than in intact rats or in testosterone-treated castrated rats. The activity of hepatic uridine diphosphate (UDP)-glucuronyltransferase, the enzyme catalyzing the glucuronidation of clofibrate, was measured using either bilirubin or 4-methylumbelliferone as substrates and was found to be unaffected by castration or testosterone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Peroxisomes are subcellular organelles found in all eukaryotic cells. In the liver they are usually round and measure about 0.5-1.0 microns; in rodents they contain a prominent crystalloid core, but this may be absent in newly formed rodent peroxisomes as well as in human peroxisomes. A major role of the peroxisomes is the breakdown of long-chain fatty acids, thereby complementing mitochondrial fatty-acid metabolism. Many chemicals are known to increase the number of peroxisomes in rat and mouse hepatocytes. This peroxisome proliferation is accompanied by replicative DNA synthesis and liver growth. No clear structure-activity relationships are apparent. Many of these peroxisome proliferators contain acid functions that can modulate fatty acid metabolism. Two mechanisms have been proposed for the induction of peroxisome proliferation. One is based on the existence of one or several specific cytosolic receptors that bind the peroxisome proliferator, facilitating its translocation to the cell nucleus and the activation of the expression of specific genes. The second, perhaps more general, hypothesis involves chemically mediated perturbation of lipid metabolism. These two hypotheses are not mutually exclusive. Many peroxisome proliferators have been shown to induce hepatocellular tumours, despite being uniformly non-genotoxic, when administered at high dose levels to rats and mice for long periods. Three mechanisms have been proposed to explain the induction of tumours. One is based on increased production of active oxygen species due to imbalanced production of peroxisomal enzymes; it has been proposed that these reactive oxygen species cause indirect DNA damage with subsequent tumour formation. In rodents, an alternative mechanism is the promotion of endogenous lesions by sustained DNA synthesis and hyperplasia. Thirdly, it is conceivable that sustained growth stimulation may be sufficient for tumour formation. Marked species differences are apparent in response to peroxisome proliferations. Rats and mice are extremely sensitive, and hamsters show an intermediate response while guinea pigs, monkeys and humans appear to be relatively insensitive or non-responsive at dose levels that produce a marked response in rodents. These species differences may be reproduced in vitro using primary culture hepatocytes isolated from a variety of species including humans. The available experimental evidence suggests a strong association and a probable casual link between peroxisome-proliferator-elicited liver growth and the subsequent development of liver tumours in rats and mice. Since humans are insensitive or unresponsive, at therapeutic dose levels, to peroxisome-proliferator-induced hepatic effects, it is reasonable to conclude that the encountered levels of exposure to these non-genotoxic agents do not present a hepatocarcinogenic hazard to humans.(ABSTRACT TRUNCATED AT 400 WORDS)
Twenty-nine thioester compounds were synthesized to test their effectiveness as surrogate substrates for the insect enzyme, juvenile hormone esterase (JHE). Substrates were designed that resembled the endogenous substrate juvenile hormone (JH), with one common factor being a thioester instead of carboxyl ester found in JH. The principle of the spectrophotometric assay is based on a modification of Ellman's method. Characterization of the substrates showed that replacement of the carbon atom by a sulfur or oxygen beta to the carbonyl of the acyl group of the substrates resulted in an approximate five- to sixfold increase in the rate of hydrolysis by JHE. The specific activities of JHE, porcine liver carboxylesterase, and acetylcholinesterase were determined for the surrogate substrates. While JHE and porcine liver carboxylesterase hydrolyzed several of the substrates, acetylcholinesterase did not produce any detectable hydrolysis of the substrates. Michaelis-Menten kinetic parameters of the surrogate substrates when compared to a previously reported partition assay, utilizing radiolabeled [3H]JH III, indicated that the surrogate substrates have lower affinity as indicated by higher Km values but are more easily hydrolyzed (Vmax) by JHE. Furthermore, optimal reaction conditions for substrate hydrolysis and the spectrophotometric reaction were determined. In addition, first order rate constants for base hydrolysis and critical micelle concentrations were determined for several surrogate substrates. The spectrophotometric assay was also compared with a Vmax and research spectrophotometer, and these two instruments produced almost identical slopes. The relative potency of four transition state inhibitors of JHE was found to be similar with those of the surrogate substrates and the [3H]JH III substrate.
Thirty carbonates, thiocarbonates, carbamates, and carboxylic esters of -naphthol, -naphthol, and p-nitrophenol were synthesized and tested as substrates for liver carboxylesterases from the crude microsomal fractions of human and mouse, and purified isozymes, hydrolases A and B, from rat liver microsomes. The carbonates, thiocarbonates, and carboxylic esters of -naphthol were cleaved more rapidly than the corresponding -naphthol isomers by the mammalian liver esterases. -Naphthyl esters of acetic, propionic, and butyric acids were among the best substrates tested for these enzymes. The majority of the substrates was consistently hydrolyzed at higher rates by hydrolase B compared with hydrolase A, although the Michaelis–Menten constant (K
m) values of selected substrates differed widely with these two isozymes. Malathion was a 15-fold better substrate for hydrolase B than for hydrolase A. Compared with the corresponding carboxylates, the carbonate moiety of - and -naphthol and p-nitrophenol lowered the specific activities of the enzymes by about fivefold but improved stability under basic conditions. The optimum pH of mouse liver esterase with the acetate, methylcarbonate, and ethylthiocarbonate of -naphthol was between pH 7.0 and pH 7.6. Human and mouse liver microsomal esterase activities were about five orders of magnitude lower than the esterase activities of purified rat liver hydrolase B. A relationship between the catalytic activity of the enzymes and the lipophilicity of the naphthyl substrates indicated that (i) in the - and -naphthyl carbonate series, an inverse relationship between enzyme activity and lipophilicity of the substrates was observed, whereas (ii) in the -naphthyl carboxylate series, an increase in enzyme activity with increasing lipophilicity of the substrates up to a log P value of about 4.0 was observed, after which the enzyme activity decreased.
Peroxisome proliferators and cancer: Mechanisms and implications An overview of peroxisome proliferator-induced hepatocarcinogenesls
- Reddy
- Ms
Reddy JK and Rao MS, Peroxisome proliferators and cancer: Mechanisms and implications. Trends Pharmacol Sci 7: 438443, 1986. li. Rao MS and Reddy JK, An overview of peroxisome proliferator-induced hepatocarcinogenesls. Environ Health Perspect 93: 205-209, 1991.
Solubilization and structure-activity relationships for substrates and inhibitors of mammalian liver microsomal carbox-ylesterases
- Huang Tl
- T Uematsu
- T Shiotsuki
- B Borhan
- Li
- Bd
Huang TL, Uematsu T, Shiotsuki T, Borhan B, Li QX and Ham-mock BD, Solubilization and structure-activity relationships for substrates and inhibitors of mammalian liver microsomal carbox-ylesterases. Phurrn Res, in press.
Sex-dependent expression and clofibrate inducibility of cytochrome P450 4A fatty acid
- Sundseth
- Ss
- Waxman
- Dj
Sundseth SS and Waxman DJ, Sex-dependent expression and clofibrate inducibility of cytochrome P450 4A fatty acid
Sex-dependent expression and clofibrate inducibility of cytochrome P450 4A fatty acid ω-hydroxylases
- Sundseth
Differential regulation of soluble epoxide hydrolase by clofibrate and sexual hormones in the liver and kidneys of mice
- Pinot
Solubilization and structure-activity relationships for substrates and inhibitors of mammalian liver microsomal carboxylesterases
- T L Huang
- T Uematsu
- T Shiotsuki
- B Borhan
- Li Qx
- B D Hammock