Impact of cytochrome P450 system on lipoprotein metabolism. Effect of abnormal fatty acids (3-thia fatty acids)

Pharmac.Ther.Vol. 61, pp. 345-383, 1994

Copyright© 1994ElsevierScienceLtd Printed in Great Britain. All rights reseawed 0163-7258/94$26.00

Pergamon

Specialist Subject Editor: C. R. SIRTORI

IMPACT OF CYTOCHROME P450 SYSTEM ON LIPOPROTEIN METABOLISM. EFFECT OF ABNORMAL FATTY ACIDS (3-THIA FATTY ACIDS) ROLF K . BERGE*,~ a n d ERLEND HVATTUM~

*University of Bergen, Department of Clinical Biology, Division of Clinical Biochemistry, Haukeland Hospital, 5021 Bergen, Norway t University of Oslo, Blindern, Institute of Medical Biochemistry, 0317 Oslo, Norway Abstract--Fatty acid co-hydroxylation, peroxisomal and mitochondrial fatty acid oxidation and related lipid-metabolizing enzymes are constitutive activities of mammalian cells. The past 5 years have witnessed an increased interest in the modulation of these pathways and functions by a new group of abnormal fatty acids (sulfur-substituted fatty acid analogs), due to the metabolic and nutritional aspects related to human health and disease, and possible treatment of certain inherited peroxisomal and mitochondrial disorders. The purpose of this review is to present an overview of current knowledge in the field and to provide an account of recent developments, particularly with respect to the chemical nature of the biologically active factors and their possible mechanism of action. Keywords---Sulfur-substituted fatty acid analogs, co-hydroxylation, mitochondrial and peroxisomal t-oxidation, hypolipidaemia, cell transformation.

CONTENTS 1. Introduction 2. Metabolism of Xenobiotics 2.1. Oxygenation of xenobiotics by cytochrome P450 2.2. Oxygenation of xenobiotics by microsomal flavin-containing monooxygenase (FMO) 3. Metabolism of the Sulfur-substituted Fatty Acid Analogs 3.1. Metabolism of 3-thia fatty acids 3.2. Sulfur oxygenation 3.3. Metabolism of 4-thia fatty acids in rive 3.4. Metabolism of 4-thia fatty acids in mitochondria 3.5. Metabolism of 4-thia fatty acids in isolated hepatocytes 4. The Effects of 3-Thia Fatty Acids (Non-fl-oxidizable) and 4-Thia Fatty Acids (fl-Oxidizable) in Rats 4.1. Pleiotropic responses in the liver 4.1.1. Effects on liver weight and liver lipids 4.1.2. Effects on mierosomes 4.1.3. Effects on peroxisomes 4.1.4. Effects on mitochondria 4.1.5. Effects on cytosolic enzymes

346 347 348 348 349 349 351 351 351 353 353 353 354 354 356 359 360

:l:Corresponding author.

Abbreviations: ACO, acyl-coenzyme A oxidase; CoA, coenzyme A; DEHP, di-(2ethylhexyl)phthalata; FMO, flavin monooxygenase; HDL, high density lipoprotein; LDL, low density lipoprotein; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-response element; VLDL, very low density lipoprotein. 345

346

R.K. BERGEand E. HVATTUM 360 361 366 366

5. Mechanisms of Hypolipidemia 5.1. Triglyceride-ioweringeffect 5.2. Phospholipid-loweringeffect 5.3. Cholesterol-loweringeffect 6. Mechanisms of Modulation of Peroxisomal Biogenesis and P4504A1 Functions by 3-Thia Fatty Acids 6.1. Transcriptional regulation of genes encoding the peroxisomal fl-oxidation enzymes and P4504AI 6.2. The chemical nature of the 3-thia fatty acids and metabolic requirements for activators of the PPAR 7. Effects on Cultured Cells vs Experimental Animals 8. Carcinogenicity of Sulfur-substituted Fatty Acid Analogs 8.1. Cell transformation 8.2. Hepatocarcinogenesis 9. Concluding Remarks and Future Aspects References

367 368 369 372 373 373 374 375 376

1. INTRODUCTION Fatty acid og-hydroxylation, peroxisomal and mitochondrial E-oxidation and related lipidmetabolizing systems are constitutive activities of most mammalian cells (Van Veldhoven and Mannaerts, 1985; Rodricks and TurnbuU, 1987; Nemali et al., 1988; Vameq, 1990). These lipid-metabolizing systems are subject to modulation response to altered nutritional and physiological status. In addition, they are also modulated by different xenobiotics, generally called peroxisomal proliferators, which include hypolipidemic drugs, phthalate esters, halogenated hydrocarbons and abnormal fatty acids. The peroxisomal proliferators increase transcriptional activity of peroxisomal and cytochrome P450 genes, notably those related to fl-oxidation and co-hydroxylation. An additional mechanism of regulation (i.e. by a decreased enzyme degradation) has been found for the peroxisomal fatty acyl-coenzyme A oxidase (ACO) and cyto-chrome-P450IVA1, which is the isoform of cytochrome P450 involved in fatty acid ~o-hydroxylation. A new group of abnormal fatty acids, where a sulfur atom replaces a methylene group in the 3- or 4-position of the carbon chain of an ordinary fatty acid (3- and 4-thia fatty acids), has been presented recently (Fig. 1). The 3-thia fatty acids induce hypolipidemia and peroxisomal proliferation. They are non-E-oxidizable, but are, in other aspects, metabolized like ordinary fatty acids. The 3-thia fatty acids have been found to be excellent models for studying the response on lipid-metabolizing enzymes to rapid intracellular changes of fatty acids (abnormal fatty acids) concentrations. An overview is presented here of the current knowledge and hypotheses concerning the (~es.ical structure

N a ~ of ~

3-th!~ carbo~lic acid u% -u% - (c~ h 2-s-c~ -coo~

~ylthioaostic

c~ - ~ - ( u % )~ 0 - s - ~ - o x ~

Do4~ithioaoetlc acid

acid

1~=~ylthio~qoionic acid

FIG. 1. Structural formulas and names of synthesized sulfur-substituted fatty acid analogs. A'"'CThese fatty acids are, in common, called 3-thia fatty acids.

Impact of cytochrome P450 system on lipoprotein metabolism

347

modulation of the lipid-metabolizing enzymes by peroxisomal proliferators with special emphasis on the effects of abnormal fatty acids (i.e. thia fatty acids). The hypolipidemic effect of the abnormal fatty acids is also discussed. The present data suggest that the hypotriglyceridemic and hypocholesterolemic properties of sulfur-substituted fatty acid analogs are primarily due to the effects on the synthesis of triacylglycerol and cholesterol. The past 15 years have witnessed an increased interest in the modulation and functions of cytochrome P450, with specificity toward co-hydroxylation of laurie acid, due to its metabolic aspects related to human health and disease. The physical and catalytic properties, induction and multiplicity of cytochrome P450 have been reviewed (Lu and West, 1980). The highly favored fatty acid specific co-hydroxylations are catalyzed largely by less specific, drug-metabolizing forms of cytochrome P450 and can be induced, for example, by phenobarbital (Bj6rkhem and Danielson, 1970; Okita and Masters, 1980). The first definitive report of a cytochrome P450 species induced by a hypolipidemic agent with substrate specificity toward co- and (co-1)-hydroxylated fatty acids, namely laurie acid, appeared in 1982 (Gibson et al., 1982). The cytochrome P450 fatty acid, co-hydroxylases (P4504A1) and related lipid-metabolizing enzymes, including peroxisomal and mitochondrial fl-oxidation, are constitutive activities of mammalian cells. It is thought that the major physiological role of cytochrome P4504A1 is to mediate the metabolism of endogenous substrates (namely, fatty acids, such as arachidonic acid) in the liver. Moreover, it is suggested that the majority of hypolipidemic drugs as a class induce, alongside other cellular responses such as an increase in peroxisome number and proliferation of endoplasmic reticulum, a cytochrome P450 with specificity toward co- and (co-1)-hydroxylation of lauric acid, whcih may or may not contribute to the hypolipidemic effect exhibited. To examine the hypothesis that all structurally unrelated hypolipidemie drugs act because they are difficult to metabolize, particularly by mitochondrial fl-oxidation, we have synthesized a series of sulfur-substituted fatty acids. Tetradecylthioacetic acid is a fatty acid analog in which a sulfur atom substitutes the 3-methylene group of a normal saturated fatty acid (Fig. 1). The analog has many of the biochemical and biophysical properties of a fatty acid (Berge et aL, 1989a,b; Skrede et al., 1989; Hvattum et aL, 1992; Skorve et al., 1990a,b; Hovik et al., 1990). The purpose of this article is to present an overview of the role of the cytochrome P450 isoenzymes and flavin-containing monooxygenase (FMO), regarding the metabolism of xenobiotics, particularly the metabolism of the sulfur-substituted fatty acid analogs. In addition, we will discuss the hypolipidemic and peroxisome proliferating effects of these analogs and the underlying mechanism of action.

2. METABOLISM OF XENOBIOTICS An organism needs systems to metabolize the large numbers of potentially harmful xenobiotics, such as drugs and environmental pollutants, to which it is exposed. The metabolism of xenobiotics can be classified into 'phase I' metabolism, in which the substrate is oxygenated, and 'phase II', in which the enzymes use the oxygen as a site for further metabolism (e.g. glucuronidation, and sulfate, glutathione or glycine conjugation). Detoxification usually requires both phase I and phase II enzymes. The metabolism of these compounds (resulting in their detoxification or, in some cases, activation to reactive intermediates capable of eliciting toxic, teratogenic or mutagenic effects) is critical to our ability to respond to exposure to foreign chemicals. In addition to their actions as substrates, many xenobiotics themselves act to regulate phase I and phase II drug metabolism by causing an increase in the levels of these enzymes. Only phase I metabolism will be discussed in this review.

348

R . K . BEROE and E. HVATTUM 2.1. OXYGENATIONOF XENOBIOTICSBY CYTOCHROME P450

Monooxygenases are capable of carrying out a myriad of chemical reactions. The common thread of all oxidative reactions is the insertion of one atom of atmospheric oxygen into the substrate, often producing a highly unstable intermediate, which breaks down to a final product(s). What is known to be the cytochrome P450 gene superfamily encodes numerous enzymes that are remarkable in the variety of chemical reactions catalyzed and in the number of substrates attacked (Ryan and Levin, 1990; Black and Coon, 1987; Guengerich, 1991). The substrates range from chromate, carbon disulfide and ethanol to steroids and 5-ring polycyclic aromatic hydrocarbons (Nebert and Gonzalez, 1987). The catalytic activity of mammalian P450 drug-metabolizing enzymes is located in the endoplasmic reticulum and inner mitochondrial membrane (Black and Coon, 1987; Nebert et al., 1982). Most of the reactions begin with the transfer of electrons from NADPH or NADH to either NADPH-cytochrome P450 reductase in the microsomal system or a ferridoxin reductase and a non-heme iron protein in the mitochondrial and bacterial systems and then to cytochrome P450. This leads to the reductive activation of molecular oxygen, followed by the insertion of one oxygen atom into the substrate (see for example Porter and Coon, 1991). Consonant with the multiplicity of P450s is the considerable diversity in the regulation of these enzymes. They can be regulated at levels of transcription, processing, mRNA stabilization, translation and enzyme stability by a number of different inducing agents (Gonzalez, 1989). The fatty acid ¢,-hydroxylases, localized in the endoplasmic reticulum (microsomes), belong to the cytochrome P450 gene superfamily, as based on the sequence identity with other cytochrome P450 enzymes. The biological oxidation of fatty acids at the og-carbon atom was first reported by Verkade et al. (1933). The co-hydroxylases are historically important as the first cytochrome P450 enzymes to have been solubilized and partially purified (Lu and Coon, 1968), and this brought the cytochrome P450 field into the era of modern biochemistry. The co-hydroxylases oxidize the terminal methyl groups of saturated and unsaturated fatty acids, including derivatives, such as prostaglandins, thromboxanes and prostacyclins (Kupfer, 1980). The genes for several of the ~o-hydroxylases have been cloned. These include, for example, the genes for rat liver and kidney lauric acid ¢o-hydroxylases (cytochromes P4504A1, 4A2 and 4A3) (Hardwick et al., 1987; Kimura et al., 1989a,b). The best characterized of this family of enzymes is the cytochrome P4504A1. This enzyme is a 58,222 Da protein that represents approximately 1-2% of the cytochrome P450 in uninduced rat liver microsomes and 16-30% after induction with clofibrate (CaJacob et al., 1988). The cytochrome P4504A 1 exhibits a high preference for hydroxylation at the terminal (o~) methyl group over the internal (co-n) methylene groups (CaJacob et al., 1988). The fatty acid ¢o-l-hydroxylations are catalyzed largely by less specific, drug-metabolizing forms of cytochrome P450 (Tanaka et al., 1990; Romano et al., 1988; Falck et al., 1990). The biological roles of the co-hydroxylases, in addition to their possible involvement in accelerating the catabolism and excretion of lipophilic fatty acids and fatty acid derivatives, are poorly understood. However, evidence suggests that some cytochrome P450 og-hydroxylation products (i.e. a~-hydroxylated arachidonic acid) have significantly physiological activities and may possibly play a role in processes such as hypertension (Escalante et al., 1991; Kauser et al., 1991). The administration of clofibrate and many other hypolipidemic agents to rodents results in an increase in the transcription of genes for proteins involved in peroxisomal/~-oxidation and in the morphologically detectable proliferation of peroxisomes (Reddy and Lalwani, 1983; Reddy et al., 1986; Hawkins et al., 1987). The same compounds simultaneously enhance the transcription of the gene for cytochrome P4504A1 (Hardwick et al., 1987). The correlation between the ability of peroxisomal proliferators to induce both cytochrome P4504A1 and peroxisomal /~-oxidation suggests the possibility of a cause and effect relationship between the two phenomena (Sharma et al., 1988; Lake et al., 1984). This is, however, still uncertain, and will be discussed in Section 6.

2.2. OXYGENATIONOF XENOBIOTICSBY MICROSOMALFLAVIN-CONTAININGMONOOXYGENASE(FMO) In addition to the cytochrorne P450 enzymes, mammalian tissue also contains a flavoprotein that catalyzes NADPH- and oxygen-dependent oxidation of a wide variety of xenobiotics. NADPH-de-

Impact of cytochrome P450 system on lipoprotein metabolism

349

pendent FMOs have been isolated from several different animals (for a review, see Ziegler, 1988). They all contain one mol of flavin adenine dinucleotide per monomer and make up about 1% of the protein of the homogenate and more than 3% of the microsomal protein in liver from female hogs (Dannan and Guengerich, 1982). The catalytic mechanism is known in some detail only for the hog liver enzyme. NADPH binds to the enzyme and reduces the flavin and dioxygen reacts with the reduced flavin, forming a stable 4ct-hydroperoxyflavin intermediate. Substrate binds and is oxygenated by oxygen transfer from the hydroperoxyflavin to substrate (Poulsen and Ziegler, 1979; Beaty and Ballou, 1980, 1981a,b). The monooxygenase catalyzes the oxygenation of a wide variety of xenobiotics. Compounds containing amines, hydroxylamines and hydrazines are usually excellent substrates for the hog liver monooxygenase (for a review, see Ziegler, 1988). The FMO also catalyzes the oxygenation of a wide range of functional groups bearing a sulfur atom (Taylor and Ziegler, 1987; Ziegler, 1980, 1988). The high relative concentration and loose substrate specificities of FMOs in mammalian tissues suggest that these enzymes contribute substantially to the oxidative metabolism of xenobiotic nucleophiles. Activity in some tissues is under hormonal control, but the enzyme is not induced by xenobiotics. 3. METABOLISM OF THE SULFUR-SUBSTITUTED FATTY ACID ANALOGS We have synthesized a series of sulfur-substituted fatty acids and suggested a hypothesis that all structurally unrelated peroxisomal proliferators act because they are difficult to metabolize, particularly by mitochondrial fl-oxidation. Tetradecylthioacetic acid is a fatty acid analog in which a sulfur atom substituted the 3-methylene group of a normal saturated fatty acid (Fig. 1). The analog has many of the biochemical and biophysical properties of a fatty acid (Berge et al., 1989a,b; Skrede et al., 1989; Skorve et al., 1990a, b; Hovik et aL, 1990). For comparison, we have also synthesized tetradecylthiopropionic acid, which, presumably, may undergo one cycle of fl-oxidation as the sulfur atom substitutes the 4-methylene group of a normally saturated fatty acid. The 3-thiadicarboxylic acid is a fatty acid in which the 3-methylene groups in both ends of the symmetrical molecule are substituted by sulfur atoms. 3.1. METABOLISMOF 3-THIA FATTY ACIDS

The thia fatty acid analogs containing a sulfur in the 3-position (3-thia fatty acids) cannot be fl-oxidized (Lau et al., 1988). The only methods of metabolizing the 3-thia acids are, consequently, co-oxidation to a dicarboxylic acid, followed by fl-oxidation from the co-end. Studies on the oxidation of dicarboxylic acids in mitochondria and peroxisomes suggest that the peroxisomes, as well as the mitochondria, can participate in the oxidation of these compounds (Draye et al., 1988; Pourfarzam and Bartlett, 1991). However, there is strong evidence that the peroxisomes are the main site for oxidation of medium-chain dicarboxylic acids in the liver (Bergseth et al., 1990b; Cerdan et al., 1988; Suzuki et al., 1989; Poosch and Yamazaki, 1989). The 3-thia fatty acid, dodecylthioacetic acid, has been shown to be excreted as short sulfoxy dicarboxylic acids in the urine of rats after intraperitoneal injection of the radioactive analog (Bergseth and Bremer, 1990). The major product was carboxypropylsulfoxyacetic acid, and some bis(carboxymethyl)sulfoxide was also found. Carboxypropylsulfoxyacetic acid was also the major metabolite when incubating isolated rat hepatocytes with dodecylthioacetic acid (Bergseth et al., 1990a). In addition to sulfur oxygenation, the 3-thia fatty acid analog evidently must have been co-hydroxylated and oxidized to a dicarboxylic acid before being chain shortened by fl-oxidation from the co-end (Fig. 2). When studying the initial metabolism of dodecylthioacetic acid, three main products were found in short-time incubations with normal rat liver microsomes (Hvattum et al., 1991). They were identified as co- and (co-1)-hydroxylated dodecylthioacetic acid and sulfur-oxygenated dodecylthioacetic acid. They were formed at about equal rates. In longer time experiments, a fourth product was detected, which was identified as co-hydroxylated and sulfur-oxygenated dodecylthioacetic acid, i.e. co-hydroxydodecylsulfoxyacetic acid. Time-curve experiments demonstrated that this JPT 61/~-E

350

R . K . BERGE and E. HVATTUM

CH3-CH2--(CH2)t2-S-CHz.-COOH

\

/

S-oxygenation

~-hydroxylation

CIt3--CH2--(CH2)I2"-S--CtI~CO~

OIt-~Cltf--(CH2)n--S-CH2-~Ott

I

J

S-oxygenation

-,,.

eo--hyd.roxylation

o/ Jl

O a'-- f~-qz~I 3-CH2-" (OH2) 12"-S- C~.-[2-(~O(~[-I

I

m-oxi~on ¢Z'~tosol

HOOI-P-~3-CIt2--(a-I2)n--S-CIt~CA~H

FIG. 2. Schemeto show the proposed first steps in the metabolism of tetradecylthioacetic acid in rat liver microsomes. Modified from Hvattum et al. (1991).

product was formed from both co-hydroxylated dodecylthioacetic acid and sulfur-oxygenated dodecylthioacetic acid. The co-hydroxylation of dodecylthioacetic acid was almost completely inhibited by carbon monooxide and significantly reduced by lauric acid, the preferred substrate for the co-hydroxylation system (Hvattum et al., 1991). The activity increased in liver microsomes from rats treated with different peroxisome proliferators, tiadenol, 3-thiadicarboxylic acid and tetradecylthioacetic acid (so-called adapted rats). This indicates that dodecylthioacetic acid is ~o-hydroxylated by the same enzyme system that catalyzes co-hydroxylation of lauric acid (Kusunose et al., 1981), i.e. the inducible cytochrome P4504A1. When comparing the specific activity of co-hydroxylation of dodecylthioaeetic acid with that of lauric acid, the two were equal in both normal and adapted rat liver microsomes, indicating that dodecylthioacetic acid was as good a substrate as lauric acid. In contrast to co-hydroxylation, sulfur oxygenation of dodecylthioacetic acid was not induced by tetradecylthioacetic acid (Hvattum et al., 1991). Interestingly, this alters the metabolic products of dodecylthioacetic acid in isolated hepatocytes from adapted rats. The main metabolic product from these cells is shifted from carboxypropylsulfoxyacetic acid to a shorter dicarboxylic acid in which the sulfur atom is not oxygenated, i.e. bis(carboxymethyl)sulfide (Bergseth et al., 1990a). Evidently, the increased hydroxylation capacity in adapted rat hepatocytes channels most of the intermediates directly to co-oxidation without a prior sulfur oxygenation. In addition, the resulting dicarboxylic acids are B-oxidized to the shortest dicarboxylic acid possible, probably due to an increased capacity of the peroxisomal //-oxidation system.

Impact of cytochrome P450 system on lipoprotein metabolism

351

3.2. SULFUROXYGENATION The sulfur oxygenation of dodecylthioacetic acid was shown to be catalyzed by liver microsomal FMOs (Hvattum et al., 1991). The reaction was dependent on either NADH or NADPH with a Km (NADH)/Km (NADPH) ratio of nearly 5. This is in agreement with a previous report on the purified hog liver FMO enzyme, which is shown to be active with both NADH and NADPH with a Kd ratio of approximately 20 (Beaty and Ballou, 1981a, b). Inhibition studies showed that sulfur oxygenation was not reduced by carbon monooxide, but markedly reduced by methimazole (Hvattum et al., 1991), a sulfur-containing compound, which is exclusively oxidized by FMO (Ziegler and Poulsen, 1978). FMO's ability to catalyze sulfur oxygenation of compounds containing carboxylic groups is, however, questionable. Investigations have shown that purified hog liver FMO does not catalyze sulfur oxygenation in molecules with a carboxylic group one or two carbons removed from the sulfur atom (Taylor and Ziegler, 1987). In contrast, S-benzyl-L-cysteine, which has a carboxyl group two carbons removed from the sulfur atom, was sulfur oxygenated by FMO in rat liver microsomes (Sausen and Efarra, 1990). The metabolism of tiadenol, a sulfur-containing hypolipidemic agent, has also been studied, and sulfoxide and sulfone carboxylic acids were detected (Maffei-Facino et al., 1986). The oxygenation of the sulfur atom in this study was suggested to be catalyzed by a cytochrome P450 isoenzyme (Maffei-Facino et al., 1986). FMO activity was not detected in this study, possibly because the enzyme solution was preincubated at 37°C without the addition of NADPH. This will inactivate the FMO (Ziegler, 1988). 3.3. METABOLISMOF 4-THIA FATTY ACIDS IN VIVO

The long-chain thia fatty acids with the sulfur in the 4-position are fl-oxidizable (Lau et al., 1988, 1989). When tetradecylthiopropionic acid and tetradecylsulfoxypropionic acid are injected intraperitoneally into rats, they are both metabolized to short-chain dicarboxylic sulfoxides excreted in rat urine (Hvattum et al., 1992), mainly as carboxypropylsulfoxypropionic acid and, in addition, some carboxymethylsulfoxypropionic acid. This indicates that tetradecylthiopropionic acid and tetradecylsulfoxyopropionic acid are metabolized similarly to dodecylthioacetic acid, i.e. an initial to-hydroxylation and sulfur oxygenation followed by oxidation to dicarboxylic acid and chain shortening from the co-end, probably in the peroxisomes. In addition, an unidentified product was formed from tetradecylthiopropionic acid. This was the main product 24-hr post-injection, and probably a mitochondrial fl-oxidation product, since it was not found with tetradecylsulfoxypropionic acid. Thia acids with the sulfur atom oxygenated are not activated to coenzyme A (CoA) esters (Aarsland and Berge, 1991) and, therefore, non-fl-oxidizable (Fig. 3). 3.4. METABOLISMOF4-THIA FATTYACIDSIN MITOCHONDRIA In isolated mitochondria, tetradecylthiopropionic acid was metabolized to two main products. One was identified as malonic semialdehyde. This is in agreement with a previous report on the oxidation of 4-thia acids (Lau et al., 1989), where the 4-thia-trans-2-alkenoyl-CoA derivative is slowly hydrated by purified enoyl-CoA hydratase, and the corresponding thiohemiacetal fragments spontaneously to malonylsemialdehyde-CoA and an alkylthiol. The other product from tetradecylthioacetic acid proved to be a thiohemiacetal formed in a spontaneous reaction between malonic semialdehyde and CoA. Malonic semialdehyde was also shown to react spontaneously with other compounds containing free thiols, e.g. dithiotreitol and glutathione. Interestingly, a metabolite from ethanol, acetaldehyde, has also been shown to react spontaneously with CoA (Ammon et al., 1969). Addition ofthiol to carbonyl groups is a well-known reaction where the thiohemiacetals are more or less stable (Lienhard and Jencks, 1966; Field and Sweetman, 1969). The metabolism of malonic semialdehyde has been a subject of discussion (Griffith, 1986). Malonic semialdehyde is generated from fl-alanine by transamination (Roberts and Bregott, 1953; Phil and Fritzson, 1955; Kupiecki and Coon, 1957), probably from 3-methylthiopropionate formed in the transamination pathway of methionine (Steele and Benevenga, 1979) and from propionyl-CoA by fl-oxidation. Two different mechanisms of metabolism have been described. First, malonyl semialdehyde-CoA can be converted to malonyl-CoA (probably via malonic acid) followed by decarboxylation to acetyl-CoA (Vagelos, 1960). Second, malonic semialdehyde can be converted directly to

352

R . K . BERGE and E. HVATTUM

acetyl-CoA with the concomitant release of CO2 (Yamada and Jakoby, 1960). Later, it was shown that malonyl-CoA is not an intermediate in the metabolism of //-alanine to aeetyl-CoA (Scholem and Brown, 1983). By isolating the product from mitochondrial incubation with [1-14C] tetradecylthiopropionic acid, i.e. [1-14C]malonic semialdehyde, the metabolism of this molecular was studied in mitochondrial extract (Hvattum et al., 1992). [1-~4C]Malonic semialdehyde was metabolized to 14CO: and probably acetyl-CoA in mitochondrial extracts. This reaction was dependent on NAD and CoA and was not inhibited by malonic acid or malonyl-CoA, indicating a direct conversion of malonic semialdehyde to acetyl-CoA with the concomitant release of COs (Fig. 4). Recently, a methylmalonic semialdehyde dehydrogenase has been purified and cloned and shown to be active with both methylmalonic and malonic semialdehyde, the products being propionylCoA or acetyl-CoA, respectively, and dependent on CoA and NAD (Goodwin et al., 1989; Popov et al., 1992; Kedishvili et al., 1992). Hovik et al. (1990) found that 4-thia acids inhibit mitochondrial and peroxisomal//-oxidation in a way that suggested that an inhibitor of//-oxidation was generated during this reaction, presumably the 4 - t h i a - t r a n s - 2 - e n o y l - C o A intermediate (Hovik et al., 1990). The finding that rats develop hepatic lipidosis after treatment with 4-thia fatty acid (Berge et al., 1989a,b) correlates well with the observation that it is a strong inhibitor of mitochondrial//-oxidation. It is tempting to speculate that malonic semialdehyde, which reacts spontaneously with CoA, might indirectly inhibit mitochondrial//-oxidation through a depletion of the mitochondrial CoA pool (Fig. 4).

CH3(CH=)13-S-CH=-CH2-CO0H Q-Oxldlltlon

/

HO-CH2(CH2)IS-S-CH2-CH2-C OOH

~

Sulfuroxygenation CH3(CH2)13-SO-CH2-CH2-COOH

Sulphur oxygenation

.q-Oxidation

HO-CH2(CH2)13-SO-CH2-CH2-COO H qlCytouollcoxidation HOOC-(CH2)ls-SO-CH2-CH2C00H

I I

Peroxieomai

6-oxidation

HOOC-(CH2Is-SO-CH2-CH2COOH Peroxleomal B-oxidation

HOOC-(CH~)-SO-CH2-C2.HCOOH FIG. 3. Metabolic pathway of tetradecylthiopropionic acid. Scheme to show the metabolism

of tetradecylthiopropionic acid in rat liver extramitochondrial fractions. Modified from Hvattum et al. (1992).

Impact of cytochrome P450 system on lipoprotein metabolism

353

CHs(CH2)ls-S-CH2-CH2-COOH Synthetaae

CH3(CH2)13-S-CH2-CH2-CO-SCoA Dehydrogenase

CH3(CH2)13-S-CH=CH-CO-SCOA Hydratase

CH3(CH2)13-S-CHOH-CH2-CO-SCoA S ~ Spontaneous CH3(CH2)I$-SH + CHO-CH2-CO-SCOA Hydrolase

CHO-CH2-COOH MS-dehydrogenase

CH3-CO-S-CoA + CO2 FIG. 4. Metabolic pathway of tetradecylthiopropionic acid. Scheme to show the metabolism of tetradecylthiopropionic acid in rat liver mitochondria. Data from Hvattum et al. (1992). 3.5. METABOLISMOF 4-THIA FATTY ACIDS IN ISOLATEDHEPATOCYTES No short dicarboxylic sulfoxides were detected after incubating isolated hepatocytes with tetradecylthiopropionic acid (Hvattum et al., 1992). The HPLC-chromatogram of the acid-soluble extract showed only unidentifiable, non-specific products, probably thiohemiacetal products from malonic semialdehyde reacting spontaneously with compounds containing free thiol groups. This indicates that the main metabolism of tetradecylthiopropionic acid in isolated hepatocytes is via mitochondrial oxidation (Fig. 4). In contrast, when incubating isolated hepatocytes with the non-fl-oxidizable sulfoxide of tetradecylthiopropionic acid (tetradecylsulfoxypropionic acid), the acid-soluble metabolites were identified to be short dicarboxylic sulfoxides, mainly carboxypropylsulfoxypropionic acid and some carboxymethylsulfoxypropionic acid.

4. THE EFFECTS OF 3-THIA FATTY ACIDS (NON-fl-OXIDIZABLE) AND 4-THIA FATTY ACIDS (fl-OXIDIZABLE) IN RATS 4.1. PLEIOTROPICRESPONSES IN THE LIVER

A series of studies in rats has revealed that abnormal fatty acids, (3-thia fatty acids) and high-fat diets, notably those rich in C22 monoene fatty acids, promote rather pleiotropic responses in responding cells, most extensively in liver (hepatocytes) and some in heart (myocardial cells). A common feature for these fatty acids is that they are poor substrates in a number of enzyme reactions, including fatty acid fl-oxidation. The responses are partly different in the two tissues; however, this review will focus only on liver in vivo experiments and cultured hepatocytes. The hepatic responses of thia fatty acids have been studied extensively in the last four years, and can be subdivided into hepatomegaly, induction of cytochrome P4504A1, and enzymatic activities associated with endoplasmic reticulum, peroxisome proliferation and associated changes in enzyme composition. Alteration in mitochondrial number and structure and changes of key enzyme activities involved in triglyceride biosynthesis, lipogenesis and cholesterogenesis have also been reported. However, the relationship between these changes and hypolipidemia has yet to be clarified. It is clear that administration of hypolipidemic drugs to rodents results in an increased activity of certain peroxisomal enzymes and cytochrome P450 with respect to lauric acid

354

R.K. BERGEand E. HVATTUM

hydroxylation, and, in general, these changes in enzyme activity reflect a dual proliferation of peroxisomes and endoplasmic reticulum. In this review, we will discuss the subcellular responses in rats that have been treated with these sulfur-substituted fatty acids, focusing on the minimal structural constraint for a chemical to be classified as a peroxisome proliferator. The effect of the 3-thia fatty acids on peroxisome proliferation, induction of P450 a~-hydroxylases and induction of associated lipid-metabolizing enzymes will be discussed with reference to the hypolipidemic effect. 4.1.1. Effects on Liver Weight and Liver Lipids

An increase in liver weight was observed after feeding rats a diet containing 0.3% (w/v) 3-thia fatty acids. The 3-thiadicarboxylic acid, which is non-fl-oxidizable and non-og-oxidizable, was considerably more effective than tetradecylthioacetic acid (only non-fl-oxidizable) in inducing enlargement of the liver (Berge et al., 1989a,b). At a dose of 150 mg/day/kg body weight of 3-thiadicarboxylic acid, the liver weight was increased within 24 hr and continued to increase for up to 7 days, when a 1.5-fold increase was observed. Morphometric measurements of randomly selected parenchymal cells show that the two fl-blocked fatty acids generated an increase in cell volume, which included both the nuclear and the cytoplasmic volumes (Kryvi et al., 1990; Berge et al., 1989b). The changes in cell volume closely followed the changes observed in the liver weight (r = 0.82) (Kryvi et al., 1990). Whether the liver enlargement is the result of hypertrophy and/or hyperplasia is not fully elucidated. It has later been shown that the effects of 3-thia fatty acids treatment are not limited to changes in liver weights, and the analogs promote a rather pleiotropic response in rat liver. Many of the responses on proteins, lipids, proliferation of organelles and enzyme activities are closely related in terms of time- and dose-dependence (Berge et al., 1989a,b; Skorve et al., 1990a,b; Kryvi et al., 1990). These changes have been observed when analyzing total liver homogenates and isolated cellular fractions. It has been found that accumulation of protein is mostly due to proliferation of mitochondria and peroxisomes (Berge et al., 1989b). Repeated administration of these 3-thia fatty acids to normolipidemic rats results in a time- and dose-dependent decrease in liver content of triglycerides and an increase in the hepatic concentration of phospholipids (Berge et al., 1989a; Aarsland et al., 1989; Skorve et al., 1990a). In contrast, repeated administration of tetradecylthiopropionic acid increased the hepatic level of triglycerides, while the concentrations of cholesterol and phospholipids remained unchanged (Aarsland et al., 1989). The tetradecylthiopropionic acid tended to enlarge the mitochondria and did not promote proliferation of mitochondria and peroxisomes comparable to the non-fl-oxidizable fatty acid. The most striking effect of the tetradecylthiopropionic acid was the formation of numerous fat droplets in the liver cells--the volume fraction of lipid droplets increased 23-fold after tetradecylthiopropionic acid feeding (Kryvi et al., 1990). Thus, tetradecylthiopropionic acid feeding appears to cause a pathological condition associated with fat droplets and high triglyceride levels (Fig. 5). No hepatomegaly resulted subsequent to tetradecylthiopropionic acid feeding in dose- and time-course studies (Berge et al., 1989a; Asiedu et al., 1990). 4.1.2. Effects on M i c r o s o m e s Proliferation of endopolasmic reticulum in hepatocytes of rats treated with the non-fl-oxidizable S-substituted fatty acid analogs has been found to be associated with a concomitant increase in NADPH-cytochrome C (P450) reductase (Berge et al., 1989b) and cytochrome P4504A1 (unpublished data). Omega oxidation of fatty acids, i.e. oxidation of fatty acids at the terminal methylene carbon, was stimulated after repeated administration of 3-thiadicarboxylic acid and tetradecylthioacetic acid (Hvattum et aL, 1991). A close relationship between induction of microsomal og-hydroxylation of fatty acid and peroxisomal fl-oxidation was observed with a correlation value of 0.971 (Hvattum et al., 1991). The correlation found between the activity of peroxisomal fl-oxidation of palmitoyl-CoA and the microsomal on-oxidation of fatty acids supports the suggestion by Lake et al. (1984) that there is a common mechanism responsible for the initiation

Impact of cytochrome P450 system on lipoprotein metabolism

355

of both the stimulated microsomal and peroxisomal responses. In contrast, tetradecylthiopropionic acid had no effect on o-oxidation. Corresponding to the intracellular localization of the palmitoyl-CoA synthetase activities, enzymes exist that reverse the activation. It has been found that the palmitoyl-CoA hydrolase activity in the microsomes decreased in a dose- and time-dependent manner subsequent to 3-thiadicarboxylic acid and tetradecylthioacetic acid feeding. In contrast, the palmitoyl-CoA synthetase activity increased. Results presented in the report by Aarsland and Berge (1991) showed that the 3-thia fatty acids were activated to their CoA thioester derivatives in all three cellular fractions (mitochondria, peroxisomes, microsomes) where the palmitoyl-CoA synthetase is known to reside. In untreated animals, a tetradecylthioacetic acid-CoA synthetase activity, which was 50% of the corresponding palmitoyl-CoA activity, was found in the microsomal fraction. The three sulfur-substituted acids modified the palmitoyl-CoA synthetase activity during feeding. Tetradecylthioacetic acyl-CoA synthetase activity was found to run in parallel with the palmitoyl-CoA synthetase activity; either the enzyme was stimulated (tetradecylthioacetic acid and 3-thiadicarboxylic acid) or inhibited (tetradecylthiopropionic acid). These results strongly suggest that the xenobiotic tetradecylthioacetic acid and palmitate are activated by the same enzyme, i.e. microsomal long-chain acyl-CoA synthetase. Sulfur-substitution of the fatty acid seems to reduce its availability for activation by 50% or more. The 3-thiadicarboxylic acid has been shown to be considerably more potent than tetradecylthioacetic acid in causing stimulation of the glycerophosphate acyltransferase activity in the

Fro. 5. Electron micrograph of hepatocytes showing the relative sizes and numbers of fat vesicles (F) from normal rats (a) and from tetradecylthiopropionic acid-treated rats (b).

356

R. K. BERGE and E. HVATTUM

microsomal fraction, and this effect was observed within hours of feeding (Berge et al., 1989b). Tetradecylthiopropionic acid tended to decrease the glycerophosphate acyltransferase activity both in mitochondria and microsomes (Berge et al., 1989a). In a time-course study, repeated administration of 3-thiadicarboxylic acid and tetradecylthioacetic acid caused an increase (35%) of diacylglycerol acyltransferase activity after 10 days of feeding. Administration of tetradecylthiopropionic acid, however, reduced the enzyme activity by more than 80% at that time (Skorve et al., 1990b). Hepatic phosphatidate phosphohydrolase activity and CTP: phosphocholine cytidylyltransferase activities are found both in the microsomes and in the cytosol. Distribution of the enzymes between the soluble and the particulate fractions is probably an important regulatory mechanism for these enzyme activities (Brindly, 1984; Vance and Vance, 1990). It has been shown that administration of tetradecylthioacetic acid and 3-thiadicarboxylic acid cause a decrease in both microsomal and cytosolic phosphatidate phosphohydrolase activites, although to a lesser extent in the microsomal fractions (Skorve et al., 1990a). Thus, no translocation of this enzyme from the cytosolic compartment to the endoplasmic reticulum was observed after repeated administration of hypolipidemic peroxisome proliferating fatty acid analogs (Skorve et al., 1990a). Repeated administration of tetradecylthiopropionic acid resulted in an increased enzyme activity both in cytosolic and microsomal fractions (Skorve et al., 1990a). Thus, the activity of this enzyme correlates well with the amount of liver triglycerides in rats fed 3-thia fatty acids. The proliferation of peroxisomes and mitochondria observed after 3-thia fatty acid treatment would seem to require an increase in the rate of synthesis of membrane phospholipids and, hence, an increase in CTP: phosphocholine cytidylyltransferase activity. Such an increase has actually been observed after repeated administration of 3-thiadicarboxylic acid and tetradecylthioacetic acid (Skorve et al., 1990a,b). However, no translocation of this enzyme from the cytosolic compartment to the microsomes was observed. Tetradecylthiopropionic acid treatment decreased both the microsomal and cytosolic CTP:phosphocholine cytidylyltransferase activities. 4.1.3. Effects on P e r o x i s o m e s

Peroxisomes are cytoplasmic organelles widely distributed in animal cells and they may differ in size and enzymatic content from organ to organ (de Duve, 1969). Moreover, it has been found that the s-values of liver peroxisomes change after feeding rats peroxisome proliferators (Flatmark et al., 1981; Berge et al., 1984a) and high fat diets (Christiansen et al., 1981; Berge et al., 1987a,b). As most of the peroxisomal enzyme activities and metabolic pathways have their counterparts in other cellular compartments (mitochondria and endoplasmic reticulum), modulation of peroxisome biogenesis and peroxisomal enzyme activities by the action of inducers requires careful consideration of the methods used to monitor the changes. Thus, the data in this review have been obtained with specific enzyme assays and optimal subcellular fractionation and morphological procedures. The peroxisomal //-oxidation has been assayed selectively by the acyl-CoA-dependent H202 production (Small et al., 1985) and by the cyanide-insensitive acyl-CoA oxidation (Lazarow and de Duve, 1976). In the liver, comparable results have been obtained by the two methods. The ability of 3-thia fatty acids to initiate proliferation of peroxisomes has been established. At the ultrastructural level, it has been found that 3-thiadicarboxylic acid, at a dose of 150 mg/day/kg body weight, increased the size (Fig. 6) and number (Fig. 7) of peroxisomes more than 2- and 6-fold, respectively. Furthermore, the frequency of dense cores in the peroxisomes decreased from 60 to 8%. The volume fraction of the peroxisomes in the liver of 3-thiadicarboxylic acid-treated rats increased by a factor of 8 (Kryvi et al., 1990). The 3-thiadicarboxylic acid was considerably more potent than tetradecylthioacetic acid. Tetradecylthioacetic acid, at a dose of 500 mg/day/kg body weight, increased the volume fraction of peroxisomes 4.5-fold, the mean volume 1.9-fold and the number of peroxisomes 3.7-fold (Berge et al., 1989b; Kryvi et al., 1990). Thus, the dicarboxylic acid is over six times more potent than the tetradecylthioacetic acid in inducing peroxisome proliferation. These data suggest that a new population of peroxisomes is generated, and that administration of 3-thia fatty acids results in an enhanced peroxisomal 0-oxidation activity and fatty acyl-CoA oxidase activity (Berge et al., 1989b), as well as increased quantities of acyl-CoA oxidase. Peroxisomal palmitoyl-CoA hydrolase (Berge et al., 1989a,b) and several peroxisomal integral

Impact of cytochrome P450 system on lipoprotein metabolism

357

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358

R. K. BERGEand E. HVATTUM Time of exposure (hr)

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(control)

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coding for enzymes of peroxisomal fl-oxidation, e.g. fatty acyl-CoA oxidase. Rats treated with 3-thiadicarboxylic acid and tetradecylthioacetic acid showed a significant increase of acyl-CoA oxidase m R N A 8 hr after feeding and at 12 hr a 6-fold increase was observed (Fig. 8). At 24-hr-feeding, however, the fatty acyl-CoA oxidase m R N A was almost normalized to the basal value. The fatty acyl-CoA oxidase enzyme activity continued to increase from 12 to 24 hr after feeding (Fig. 9). This suggests that there may be two different inductive mechanisms operating, one at the level of m R N A and the other affecting the stability of the acyl-CoA oxidase enzyme, i.e. the half-life of acyl-CoA oxidase enzyme may be increased in livers of rats fed sulfur-substituted fatty acid analogs. A similar effect has been observed with partially hydrogenated fish oil (Horie and Suga, 1989, 1990). It has also been found that 3-thiadicarboxylic acid and tetradecylthioacetic acid increased the

Impact of cytochrome P450 system on lipoprotein metabolism

359

catalase activity, whereas the urate oxidase activity was decreased (Berge et al., 1989a,b). The palmitoyl-CoA synthetase activity and the acyl-CoA:dihydroxyacetonephosphate acyltransferase, catalyzing the key enzymatic step in ether glycerolipid synthesis, increased in the peroxisomal fractions isolated from rat liver (Skorve et al., 1990b). Tetradecylthiopropionic acid is reported to cause marginal morphological changes of peroxisomes compared with the 3-thiadicarboxylic acid and tetradecylthioacetic acid (Berge et aL, 1989b; Kryvi et al., 1990). The most striking effect of the tetradecylthiopropionic acid is an accumulation of hepatic triglycerides and increased number of fat droplets (Fig. 5). The above data strongly suggest that the potency of selected compounds as proliferators of peroxisomes depends on their susceptibility to fl-oxidation. Experimental data suggest that the minimal structural requirement for peroxisome proliferation may be (1) a carboxylic acid group linked to (2) a hydrophobic backbone with (3) poor susceptibility to fl-oxidation. It is conceivable that blockage for c~-oxidation may potentiate the peroxisome-proliferating capacity, since 3-thiadicarboxylic acid is more potent than tetradecylthioacetic acid. Notably, the stimulation of peroxisomal fl-oxidation by eicosapentaenoic acid (Aarsland et al., 1990b), but not with palmitate, conforms to the previously defined requirement for initiation of peroxisome proliferation. Increased production of liver peroxisomes accompanied by stimulated fl-oxidation activity is observed after feeding of certain high fat diets, especially diets rich in unsaturated C20-Cn fatty acids, which are relatively poorly oxidized by the mitochondrial fl-oxidation system. 4.1.4. Effects on Mitochondria

Treatment of rats with the non-fl-oxidizable sulfur-substituted fatty acids results in several observable mitochondrial changes, which include proliferation, structural changes and induction of enzymes. It has been found that administration of 3-thiadicarboxylic acid and tetradecylthioacetic acid

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Fxa. 9. Induction of fatty acyl-CoA oxidase activity after treatment with sulfur-substituted fatty acid analogs. Livers were taken from animals at the indicted times after treatment with individual fatty acid analogs. 3-Thiadicarboxylic acid (O), tetradecylthioacetic acid (A), tetradecylthiopropionic acid (A) and control (O). Acyl-CoA oxidase activity was determined in the post-nuclear fraction from three to six per group.

360

R.K. BERGEand E. HVATTUM

resulted in an increase in the total capacity for liver mitochondrial/~-oxidation and in the oxidation of palmitoyl-CoA and palmitoyl-L-carnitine by isolated liver mitochondria. At the same time, it was observed that carnitine palmitoyltransferase activity increased. In contrast, the oxidation of fatty acids by isolated mitochondria decreased by nearly 80% after tetradecylthiopropionic acid treatment (Berge et al., 1989a; Skorve et al., 1990a). Hepatic mitochondria are oval in shape, with an average area of 0.52/~m 2 and volume of 0.212 #m 3 (Kryvi et al., 1986). Repeated administration of the 3-thiadicarboxylic acid resulted in smaller mitochondria (Fig. 6), and at a dose of 150 mg/day/kg body weight, the mean volume of mitochondria was 0.324/./m 3. Proliferation of mitochondria was observed after 3-thiadicarboxylic acid feeding, with the number of mitochondria increasing 4-fold after treatment (Fig. 7). In addition to initiation of peroxisomal proliferation and induction of peroxisomal/~-oxidation enzymes, tetradecylthioacetic acid has been found to increase the number of mitochondria and their volume fraction in rat hepatocytes (Kryvi et al., 1990; Berge et al., 1989b). It has been found that the 3-thia fatty acids increased the activities of palmitoyl-CoA synthetase and palmitoyl-CoA hydrolase in the mitochondrial fractions isolated from rat livers (Berge et al., 1989a,b). The 3-thia fatty acids treatment also stimulated the mitochondrial glycerophosphate acyltransferase activity (Berge et al., 1989a). 4.1.5. Effects on Cytosolic E n z y m e s It has been found that the activity of cytosolic palmitoyl-CoA hydrolase is increased by 3-thiadicarboxylic acid and tetradecylthioacetic acid, with a time- and dose-curve similar to the increase in peroxisomal E-oxidation (Berge et al., 1984b, 1989b) and microsomal co-hydroxylation (Hvattum et al., 1991) (Fig. 10). This is a feature shared by different peroxisome proliferators, i.e. clofibrate, tiadenol and niadenate (Berge and Bakke, 1981), and by high-fat diets (Berge et al., 1987b, 1988; Berge and Thomassen, 1985). Clofibroyl-CoA hydrolase activity has been observed in the cytosolic fraction of rat liver. This activity can also be increased with administration of peroxisome proliferators (Lygre et al., 1986; Berge et al., 1987a,b).

5. MECHANISMS OF HYPOLIPIDEMIA 5.1. TRIGLYCERIDE-LOWERINGEFFECT In addition to their biochemical and morphological effects of the liver, 3-thia fatty acids usually decrease serum triglyceride, cholesterol and free fatty acid levels in the rat (Berge et al., 1989a; Skorve et al., 1990a,b) (Table 1). Plasma triglyceride levels are determined by a delicate balance between hepatic triglyceride synthesis and secretion on one hand, and plasma triglyceride clearance on the other. Thus, the observed reduction in plasma triglyceride levels during 3-thiadicarboxylic acid administration could be accomplished by retarded synthesis, reduced hepatic output, enhanced clearance or a combination of these factors. 3-Thia fatty acids are found to decrease triglyceride biosynthesis in the liver by several mechanisms (Table 1). Previous studies have shown that 3-thiadicarboxylic acid treatment stimulated both mitochondrial and peroxisomal fl-oxidation of fatty acids (75 and 450%, respectively) (Asiedu et al., 1990; Aarsland et al., 1989). The relative importance of the induced fl-oxidation in peroxisomes vs mitochondria for the overall fatty acid oxidation in the liver under 3-thiadicarboxylic acid treatment, however, remains to be established. The previous finding that the effects of 3-thiadicarboxylic acid on peroxisomal E-oxidation and plasma triglyceride levels were dissociable, depending on dose and time of treatment, indicates that the role of 3-thiadicarboxylic acid, as a peroxisomal proliferator, might not be crucial for the hypotriglyceridemic effect observed (Aarsland et al., 1989). Moreover, it should be emphasized, that mitochondria are, by far, the quantitatively dominating organelle in liver cells (Figs 5 and 6), implying that a 2-fold increase in mitochondrial oxidation might have a greater impact on the total E-oxidation of fatty acids than the 5-fold increase in peroxisomal oxidation (Table 1). 3-Thiadicarboxylic acid treatment resulted in a slight inhibition in the activities of ATP:citrate lyase and fatty acid synthetase (Table 1). However, the impact of these effects on lipogenesis, and,

Impact of cytochromc P450 system on lipoprotein metabolism

361

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rime of exposure ( h r ) Fx6. 10. Induction of co- and (o~-l)-hydroxylation of laurie acid in liver post-nuclear fraction from rats treated with sulfur-substituted fatty acid analogs (see Fig. 3). 3-Thiadicarboxylic acid (0), tetradecylthioacetic acid (A) and tetradecylthiopropionic acid (A). Liver post-nuclear fractions from rats treated with different compounds were incubated with [1-t4C]-lauric acid for 5 min. The results are given as nmol hydroxy lauric acid metabolites formed per rain per mg protein and the tabulated values represent means + SD for three to six rats in each experimental group. (A) Total hydroxylation of lauric acid (co + co.1); (B) to-hydroxylation of laurie acid and (C) (co-1)-hydroxylation of lauric acid.

consequently, triglyceride biosynthesis, is not self-evident. On the one hand, an inhibition of two o f the enzymes involved in fatty acid synthesis is consistent with a retarded lipogenesis, On the other hand, the finding that the activity o f the enzyme considered to be rate limiting in fatty acid synthesis, i.e. acetyl-CoA carboxylase, was unaffected by drug treatment argues against the contention that decreased fatty acid synthesis is of major importance for the triglyceride-lowering effect observed. During administration o f the drug, plasma free fatty acid levels decreased (Table 1),

R. K. BERGE and E. HVATTUM

362

Thus, 3 - t h i a d i c a r b o x y l i c acid t r e a t m e n t m i g h t interfere with b o t h the e x o g e n o u s a n d the e n d o g e n o u s s u p p l y o f hepatic fatty acids, affecting their availability for esterification a n d triglyceride biosynthesis. In a d d i t i o n , the observed inhibition o f p h o s p h a t i d a t e p h o s p h o h y d r o l a s e activity d u r i n g 3 - t h i a d i c a r b o x y l i c acid t r e a t m e n t (Skorve et al., 1990b) w o u l d further c o n t r i b u t e to a lower triglyceride synthetic rate. T h e parallel decrease in p h o s p h a t i d a t e p h o s p h o h y d r o l a s e activity a n d the hepatic a n d p l a s m a triglyceride c o n c e n t r a t i o n s suggests that the hypotriglyceridemic effect o f 3 - t h i a d i c a r b o x y l i c level m a y be largely due to its effect on the synthetic

TABLE 1. The Effect of 3-Thiadicarboxyfic Acid on Plasma and Hepatic Lipids and

Liver Enzymes Parameters

Non-treated rats

3-Thiadicarboxylic acid-treated rats

Plasma Triglycerides (mmol/L) Cholesterol (mmol/L) Phospholipids (mmol/L) Free fatty acids

1.21 _ 0.14 2.55 _ 0.31 1.47 + 0.16 0.84 ___0.08

0.75 --I-0.091 1.69 + 0.11L 0.72 ___0.05 0.43 +__0.071

3.27 ___0.09 6.77 _ 0.40 5.32 -I- 0.12 18.12+0.41

5.57 -F 0.33 l 3.23 _ 0.221 5.24 _ 0.09 21.10-t-0.271

4.27 _ 0.33 2.56 _ 0.68

6.83 _ 0.281 4.70 _ 0.161

Peroxisomal fl-oxidation Fatty acyl-CoA oxidase

1.56+0.18 9.33 + 0.30

7.84 + 0.6C 46.89 + 1.441

Glycerophosphate acyltransferase (nmol/min/mg protein) microsomal fraction mitochondrial fraction Diacylglycerophosphate acyltransferase (nmol/min/mg protein)

2.08 __+15 0.40 -+_0.09

9.56 ___0.251 0.85 ___0.091

2.83-1-0.17

4.15-t-0.401

12.23 + 1.45 1.68 _ 0.09

6.37 + 0.70 I 0.82 _ 0.051

Lipoprotein lipase (mU)

0.99 ___0.02

1.17 + 0.02 l

Hepatic lipase (mU) ATP-citrate lyase (nmol/min/mg protein)

0.93 _ 0.03

1.10 _ 0.02 l

12.50 _ 0.18

9.84 _ 0.64 l

Acetyl-CoA carboxylase (nmol/min/mg protein)

4.71 + 0.58

4.66 __.0.49

Fatty acid synthetase (nmol/min/mg protein)

0.15__+0.02

0.10+0.02 l

H M G CoA-reductase (pmol/min/mg protein)

1039 + 33

256 ___41 l

Cholesterol 7-cc-hydroxylase (nmol/min/mg protein)

36.2 + 2.3

17.3 + 3.31

364 __+8

277 _ 21

Liver Liver weight/body weight Triglycerides (# mol/g) Cholesterol (/~mol/g) Phospholipids 0t mol/g) Enzymes Mitochondrial fl-oxidation (nmol/min/mg protein) Paimitoyl-L-carnitine as substrate Palmitoyl-CoA as substrate

Phosphatidate phosphohydrolase (nmol/min/mg protein) Release phosphate Production of 14C-diacylglycerol

AcyI-CoA: cholesterol acyltransferase (pmol/min/mg protein)

Values are expressed as means + SE for six animals in each experimental group. ip < 0.05.

Impact of cytochrome P450 system on lipoprotein metabolism

363

TAaLE 2. Plasma Lipoprotein Levels in 3-Thiadicarboxylic Acid-treated and Control Rats Composition Triglycerides (mmol / L ) Total VLDL LDL HDL Cholesterol (mmol / L ) Total VLDL LDL HDL

Non -treated

3- Thiadicarboxylic acid-treated

Decrease during treatment

1.07 _+0.10 0.62 + 0.12 < 0.05 0.44 ___0.02

0.67 + 0.061 0.34 _+0.042 < 0.05 0.33 _+0.041

- 38% - 46% - 25%

2.32 _+0.12 0.44 _+0.06 1.22_+0.20 0.64 + 0.08

1.53 _+0.151 0.27 _+0.021 0.71 _+0.111 0.54 _+0.021

- 34% - 39% -42% - 16%

The values are given as mean + SE for six animals in each group. To convert mmol/L to mg/dL, multiply cholesterol values by 38.7 and triglyceride values by 88.5. Ip < 0.05; 2p = 0.055.

level. Also, this underscores the regulatory importance of phosphatidate phosphohydrolase in triglyceride biosynthesis (Bell and Coleman, 1980). In contrast, both glycerol 3-phosphate and diacylglycerol acyltransferase activities (Skorve et al., 1990a) (Table 1) increased when hepatic triglyceride synthesis and secretion were retarded. This argues against the initial and the last esterification steps (Rustan et al., 1988) in triglyceride being potential sites at which 3-thiadicarboxylic acid might modulate triglyceride synthesis. A major new finding in the present study was the effect of 3-thiadicarboxylic acid treatment on the lipoprotein fractions, with a decrease in both very low density lipoprotein (VLDL)-triglyceride and low density lipoprotein (LDL)-cholesterol levels (Table 2). The decrease in VLDL-triglyceride was associated with a reduction in the secretion of newly synthesized triglycerides (Fig. 11). Since V L D L is a precursor to LDL, it is conceivable that the observed reduction in V L D L secretion and plasma V L D L levels will affect L D L formation, which might contribute to the lowering of L D L cholesterol levels. It is of interest that the reduction in plasma triglyceride levels was slightly more pronounced (38%) than the decrease in the VLDL-triglyceride secretion (25%), implying that alterations in the clearance of VLDL-triglycerides might contribute to the decrease in plasma triglycerides. In agreement with this, the activities of plasma lipoprotein lipase and hepatic hpase 20 A .-I

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F1o. 11. Effect of 3-thiadicarboxylic acid (TD) on plasma triglyceride entry rate after a single intravenous injection of Triton WR 1339. The values are expressed as means + SD for 6 rats in each experimental group. To convert mmol/L to mg/dL, multiply by 88.5. Data from Skorve et al. (1993).

364

R.K. BERGEand E. HVATTUM A: Triacylglycerol

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FIG, 12. Effects of 3-thiadicarboxylic acid (I-q) and tetradecylthioacetic acid (O) on incorporation of [3H]waterinto secreted lipids (A), triacylglycerol; (B) cholesterol; (C) cholesteryl ester; (D) phospholipids; by cultured rat hepatocytes. The cells were incubated for 9 hr in the presence of 3 mCi [3H]water/mL, 0.2 mMoleic acid/0.08 mMbovine serum albumin and various concentrations of thia fatty acid analogues. Data represent means + SD for triplicate culture flasks.

were somewhat higher (10-20%) in 3-thiadicarboxylic acid-treated animals (Table 1), indicating a possible increase in the clearance potential of triglyceride-rich lipoproteins. However, no difference was found in either the chemical composition of the apolipoprotein pattern in isolated VLDL from control or 3-thiadicarboxylic acid-treated rats, arguing against any larger conformational changes that could affect their catabolism (data not shown). At present, we cannot, therefore, exclude that minor conformational changes in VLDL could be induced by 3-thiadicarboxylic acid administration, and further studies are needed to establish whether treatment with this drug affects the metabolic properties of VLDL. In hepatocytes cultured in the presence of oleic acid, incorporation of [3H]water into secreted lipids and triacylglycerols (Fig. 12) was lower than incorporation into synthesized lipids (Fig. 13) with the 3-thia fatty acids. This is especially marked in the presence of the monocarboxylic acid, tetradecylthioacetic acid. A similar phenomenon was observed with [3H]glycerol as the radioactive precursor (data not shown). This suggests that some of the hypotriglyceridemic effects of 3-thia fatty acids may arise from a reduction in biosynthesis and/or secretion of triacylglycerols. The degree of inhibition of [3H]water incorporation into triacylglycerols and phospholipids was at approximately the same level (Figs 12 and 13), indicating that the 3-thia fatty acids may affect a common step in the biosynthesis of these lipids. The incorporation of [3H]water into synthesized diacylglycerols was reduced even more than the incorporation into the other two lipid classes. As the sulfur-substituted fatty acids had only small effects on incorporation of [3H]water into cell monoacylglycerols (data not shown), these data suggest that 3-thia fatty acids decreased triacylglycerol synthesis by affecting the step before formation of diacylglycerols. The reduced incorporation into diacylglycerols, thus, may indicate that the activity of the enzyme phosphatidate phosphohydrolase could be affected. When the synthesis of lipids is reduced due to the presence of fatty acid analogs, the free fatty acids will be diverted from the esterification pathway. The level of free fatty acids in the hepatocytes

Impact of cytochrome P450 system on lipoprotein metabolism

365

treated with 3-thia fatty acids tended to decrease. This indicates that the mitochondrial fl-oxidation of fatty acids was increased, as the peroxisomal//-oxidation was unchanged in these hepatocytes. Thus, it is likely that the non-f-oxidizable fatty acid analogs reduced the availability of fatty acids for triacylglycerol synthesis due to increased mitochondrial fatty oxidation. The lack of effect on the peroxisomal f-oxidation confirms the in vivo data that the hypotriglyceridemic effect of the analogs can be dissociated from the proliferation of peroxisomes (Aarsland et al., 1990a,b). Malonyl-CoA, the product of the acetyl-CoA carboxylase reaction, is the substrate for the biosynthesis of fatty acids. It is also an inhibitor of carnitine palmitoyltransferase, the enzyme regulating mitochondrial fatty acid oxidation. Long-chain acyl-CoA inhibits malonyl-CoA synthesis by inhibiting acetyl-CoA carboxylase (Wong et al., 1984). An acute decrease in malonyl-CoA at 4-6 hr has been found in animals treated with tetradecylthioacetic acid (Skrede and Bremer, 1993); Asiedu et al., 1994). At that time the levels of long-chain acyl-CoA was increased (J. Knudsen and R. K. Berge, manuscript in preparation). A rise in cellular acyl-CoA after administration of 3-thia fatty acids could result in inhibition of the enzyme acetyl-CoA carboxylase. Reduced levels of malonyl-CoA could then relieve inhibition of carnitine palmitoyltransferase and the mitochondrial fatty acid oxidation would be stimulated (Skorve et al., 1990b). Thus, it is likely that the rate of triglyceride synthesis is controlled by coordinate regulation of

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FIO. 13. Time course for the effect of 3-thiadicarboxylic acid and tetradecylthioacetic acid on incorporation of [3H]water into cell-associated lipids (A), triacylglycerol; (B) cholesterol; (C) cholesteryl ester; (D) phospholipids; (E) free fatty acids; (F) diacylglycerol; by cultured rat hepatocytes. The cells were incubated up to 9 hr in the presence of 3 mCi [3H]water/mL, 0.2 mM oleic acid/0.08mM bovine serum albumin (control, ll); in addition, 100/~M of either 3-thiadicarboxylic acid (I-q) or tetradecylthioacetic acid (O). Data represent means + SD for duplicate culture flasks.

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366

R.K. BEROEand E. HVATTUM

the activities of mitochondrial p-oxidation and phosphatidate phosphohydrolase. The instant hypotriglyceridaemic effect observed in rats given 3-thia fatty acids can be explained by a sudden increase in mitochondrial fatty acid oxidation and a decrease in phosphatidate phosphohydrolase (Skorve et aL, 1990a). This alteration is accompanied by a reduction in the availability of substrates, i.e. fatty acids for glycerolipid synthesis, even before the 3-thia fatty acids induce peroxisomal fatty acid oxidation and ~o-oxidation. It is striking that tetradecylthioacetic acid treatment affects the rate of secretion to a greater extent than 3-thiadicarboxylic acid. Thus, it is possible that tetradecylthioacetic acid, in contrast to 3-thiadicarboxylic acid, may interfere with some steps in the secretory process, i.e. the assembly or transport of the VLDL particles in addition to the inhibition of triacylglycerol synthesis. 5.2. PHOSPHOLIPID-LOWERINGEFFECT

The question of a coordinated regulation of the synthesis of triglycerides and phospholipids was also addressed. Of interest is the observation that when phosphatidate phosphohydrolase was inhibited, the activity of CTP:phosphocholine cytidylyltransferase was stimulated (Skorve et al., 1990b; Asiedu et al., 1992). An inhibition of phosphatidate phosphohydrolase, coupled with a stimulation of the cytidylyltransferase, would accelerate phospholipid biosynthesis at the expense of retarded synthesis of triglycerides. As phosphatidylcholine synthesis is required for VLDL synthesis and secretion, changes in cytidylyltransferase activity, the rate-limiting enzyme of de novo phosphatidylcholine synthesis, could account for decreased VLDL triglyceride output. Thus, decreased activity of an esterifying enzyme, phosphatidate phosphohydrolase, and diversion to phospholipid formation are mechanisms by which 3-thia fatty acids may inhibit triglyceride and, subsequently, VLDL production. Moreover, the increased cytidylyltransferase activity (Table 1) could account for an increase of lipid components for proliferation of peroxisomes (Asiedu et al., 1992). This study has also addressed the question of translocating a metabolically inactive form of phosphatidate phosphohydrolase and cytidylyltransferase residing in the cytosol to their sites of action in membranes. No translocation of either phosphatidate phosphohydrolase or CTP :phosphocholine cytidylyltransferase was observed in rats fed the 3-thia fatty acid analog (Skorve et al., 1990b). Thus, the total phosphatidate and phosphocholine metabolism appears to be more relevant to VLDL triglyceride production and secretion than any one particular enzyme form (Asiedu et al., 1992). Altogether, it would seem that triglyceride formation and VLDL-triglyceride secretion is reduced with 3-thia fatty acid, due to increased mitochondrial fatty acid oxidation, reduced esterification and diversion to phospholipid formation. The results emphasize the importance of the availability of the substrate, i.e. fatty acid as a major determinant of the rate of triglyceride biosynthesis. Thus, the hypolipidemic properties of saturated 3-thia fatty acid analogs are primarily due to effects on the synthetic level. 5.3. CHOLESTEROL-LOWERINGEFFECT

The effects of 3-thiadicarboxylic acid on plasma cholesterol levels could be due to a number of factors. Of prime significance is the possibility that this effect could be obtained through retarded cholesterol synthesis and/or increased degradation. It is well documented that HMG-CoA reductase is rate-limiting in the synthesis of cholesterol under almost all experimental conditions, and inhibition of this enzyme has been shown to reduce plasma cholesterol levels (Grundy, 1988; Tobert et al., 1982; Reihnrr et al., 1990). Repeated administration of the 3-thiadicarboxylic fatty acid led to a 75% reduction of the HMG-CoA reductase activity. It is tempting, therefore, to suggest that the decrease in plasma cholesterol was mediated largely by an inhibition of HMG-CoA reductase. In addition to the effect on HMG-CoA reduetase, the activity of the rate-limiting enzyme in the degradation of cholesterol into bile acids, i.e. cholesterol 7-~-hydroxylase, was also depressed (52%) (data not shown). Furthermore, acyl-CoA:cholesterol acyltransferase activity was inhibited (24%) (Table 1), compatible with a decrease in cholesteryl ester formation. Recently, a possible role of cholesteryl ester for VLDL secretion was proposed (Cianflone et al., 1990). The impact of

Impact of cytochrome P450 system on lipoprotein metabolism

367

the reduction of these enzyme activities, however, might be less prominent and could be due to the reduction of cholesterol biosynthesis. Of interest is the observation that both HMG-CoA reductase and phosphatidate phosphohydrolase activities decreased during 3-thiadicarboxylic acid treatment. Previous studies have shown a link between these enzymes (A1-Shurbaji et al., 1991; Bjfrkhem and Berglund, 1987), demonstrating the importance of concerted regulation of cholesterol and triglyceride biosynthesis. Indeed, a parallel regulation of these two metabolic pathways would be of importance for the observed reduction in VLDL-secretion. Since the composition of VLDL was not affected by drug treatment, it seems likely that 3-thiadicarboxylic acid treatment results in a general reduction in VLDL production involving both cholesterol and triglycerides rather than a selective decrease in triglyceride biosynthesis. Although the total liver content of cholesterol was not reduced, in contrast to triglycerides (Table 1), compartmentalization, nevertheless, may result in decreased availability of cholesterol for VLDL synthesis. Using hepatocytes, cholesterogenesis and esterification of cholesterol was clearly inhibited by 3-thiadicarboxylic acid, while tetradecylthioacetic acid only marginally affected the incorporation of [3H]water into cholesterol and cholesteryl ester (Figs 12 and 13). In rat liver in vivo, we have found that both fatty acid analogs inhibited the activity of the rate-limiting enzyme in the biosynthesis of cholesterol HMG-CoA reductase, although not to the same extent (unpublished results). Thus, it is reasonable to assume that the inhibition of cholesterogenesis by 3-thiadicarboxylic acid is primarily due to inhibited HMG-CoA reductase activities. This inhibition may also account for the cholesterollowering effect of the fatty acid analog, as the incorporation of [3H]water into secreted cholesterol and cholesteryl ester was reduced to the same extent as the synthesis (Figs 12 and 13). Tetradecylthioacetic acid, however, did significantly reduce the incorporation of [3H]water into secreted cholesteryl ester and cholesterol especially at the highest dose used. That tetradecylthioacetic acid decreased the secretion of cholesterol and cholesteryl ester, without affecting the synthetic rate, agrees with the point that this 3-thia fatty acid may have an effect at some step in the secretion process of VLDL particles. In the rat in vivo, the two 3-thia fatty acids reduce plasma lipid levels by approximately the same extent (Aarsland et al., 1990a, b). 3-Thiadicarboxylic acid has, however, the greatest impact on the activities of key enzymes in the liver and reduces the hepatic level of triacylglycerols far more than tetradecylthioacetic acid does. This last fatty acid analog presumably has some additional effects on the hepatic secretion of triacylglycerols and cholesterol. The exact nature of this effect, however, is still not known. In summary, available data demonstrate that 3-thiadicarboxylic acid treatment of normolipidemic rats decreases plasma triglycerides, phospholipids and cholesterol. All effects seem to be primarily mediated on the synthetic level. These findings indicate that sulfursubstituted non-fl-oxidizable fatty acid analogs might be a useful tool for studying-and possibly treating--metabolic disturbances characterized by increased lipoproteinsynthesis.

6. MECHANISMS OF MODULATION OF PEROXISOMAL BIOGENESIS AND P4504A1 FUNCTIONS BY 3-THIA FATTY ACIDS Although mammalian peroxisomes are widely available structures that can differ in size and enzymatic equipment from organ to organ, they all share some common properties. In general, a qualitative comparison of the tissues indicates that the fatty-acid-metabolizingenzyme component is similar, whereas substantial quantitative differences exist in both the control and induced states. Furthermore, experimental evidence, has recently been presented that indicates that peroxisome proliferation and the induction of the fatty acid fl-oxidation enzymes are regulated separately and that de novo formation of peroxisomes involves a sequential biosynthesis of membrane and matrix proteins (Hawkins et al., 1987).

368

ROLF K. BERGE and ERLEND HVATTUM 6.1. TRANSCRIPTIONAL REGULATION OF GENES ENCODING THE PEROXISOMAL fl-OXIDATION ENZYMES AND P4504AI

We have examined the increase of hepatic acyl-CoA oxidase and P4504A1 mRNA levels and induction of the corresponding enzyme activities at various times (2-24 hr) after administration of a single dose of sulfur-substituted fatty acid analogs to rats (Figs 8, 9 and 10). The present study shows that the non-fl-oxidizable fatty acid analogs (3-thiadicarboxylic acid and tetradecylthioacetic acid) in contrast to the fl-oxidizable analog, tetradecylthiopropionic acid, increased both the acyl-CoA oxidase and the fatty acid co-hydroxylation activities (Figs 9 and 10). The dicarboxylic acid was more potent than the monocarboxylic acid in inducing these changes, confirming previous findings (Berge et al., 1989a,b; Hvattum et al., 1991). Other studies of the induction of the co/(co-1)-hydroxylation of fatty acids by different hypolipidemic peroxisome proliferating agents show, as found here, a preferential induction of co-hydroxylation over co-1-hydroxylation (Gibson et al., 1982). A differential kinetics of induction of the P4504A1 and acyl-CoA oxidase mRNAs was demonstrated after treating cultures of primary hepatocytes with structurally dissimilar peroxisome proliferators of differing potency (Bell and Elcombe, 1991). From these studies, induction of P4504AI mRNA appeared to be a very early event in the peroxisome-proliferating response, even prior to the induction of fatty acyl-CoA oxidase mRNA. The structurally related sulfur-substituted fatty acid analogs were used for analysis of induction kinetics in order to determine comparability with previous studies in vitro (Berge et al., 1989b; Asiedu et al., 1990). Our present results show a parallel increase in acyl-CoA oxidase and P4504A1 mRNA levels 8 and 12 hr after administration of 3-thiadicarboxylic acid and tetradecylthioacetic acid, respectively. The 3-thia fatty acid analogs did not cause a more rapid induction of P4504A1 compared with acyl-CoA oxidase mRNA (Fig. 8). The same observation was made for the corresponding enzyme activities (Figs 9 and 10). The correlation between the increase of P4504A1 and acyl-CoA oxidase mRNA levels and the induction of their corresponding enzyme activities is in contrast to previous findings (Bell and Elcombe, 1991). Furthermore, we also were able to differentiate the induction kinetics of acyl-CoA oxidase and P4504A1 mRNA levels on the basis of 3-thia fatty acids as peroxisome proliferators of differing potencies (Berge et al., 1989a,b). 3-Thiadicarboxylic acid caused a stronger increase in the quantity of both P4504A1 and acyl-CoA oxidase mRNAs compared with tetradecylthioacetic acid (8 and 12 hr) (Fig. 8). Thus, our present data obtained in vivo are not consistent with the differential kinetics of induction of P4504A1 and fatty acyl-CoA oxidase mRNAs demonstrated in cell culture by Bell and Elcombe (1991), employing different peroxisome proliferators. Indeed, the correlation between induction of mRNAs and their enzyme activities, at best, may reflect a coordinate rather than causative induction mechanism in which both processes, induction of P4504AI and peroxisome proliferation, respond to a common signal. Tugwood et al. (1992) recently have demonstrated that the 5' flanking region of the acyl-CoA oxidase gene contains a response element, peroxisome proliferator-response element (PPRE), that binds peroxisome proliferator-achieved receptor (PPAR), which appears to play a fundamental role in mediating the action of peroxisome proliferators. A coordinated mechanism would be consistent with the hypothesis that the hepatic changes triggered by peroxisome proliferators are responses to a general disturbance in fatty acid metabolism (Lock et al., 1989). In this respect, it is interesting to speculate whether the 5'-upstream region of the P4504A1 gene also contains a PPRE element that binds PPAR. This needs to be investigated, but it should be noted that the cyp4A6 gene contains a regulating element in the 5' flanking region that is structurally similar to the acyl-CoA oxidase gene (Muerhoff et al., 1992), further indicating a common regulating mechanism. Our results may equally well be coincidental in nature. If, however, the two genes are under the same transcriptional control, such a coordinate mechanism for the induction of peroxisome proliferation would be in contrast to the model proposed by Elcombe and coworkers. In that model, the proliferating agent induces cytochrome P4504A 1 and thereby increases the production of co-oxidized fatty acids, i.e. dicarboxylic acids, which, in turn, induce peroxisome proliferation. The consequence of Elcombe's model is that a dicarboxylic acid should induce fatty acyl-CoA oxidase mRNA and enzyme activity without any induction of cytochrome P4504A1. Adminis-

Impact of cytochrome P450 system on lipoprotein metabolism

369

tration of hexadecanedioic acid at different doses up to 750 mR/day/body weight causes no proliferation of peroxisomes and no increased peroxisomal //-oxidation (Berge et al., 1989b; Aarsland et al., 1989). In the present study, we have observed that a sulfur-substituted dicarboxylic acid and a monocarboxylic acid caused induction of P4504A1 mRNA and fatty acid co-hydroxylation with a time course of induction similar to those of acyl-CoA oxidase mRNA and enzymatic activity, and which was related to the potency of the peroxisome proliferator. Recently, it has been reported that fatty acids activate the PPAR, but they do not appear to be bound to this nuclear receptor themselves (G6ttlicher et al., 1992; Issemann and Green, 1990) (see also below). Tetradecylthioacetic acid was as potent as the strong peroxisome proliferator WY 14.643 in PPAR activation, whereas tetradecylthiopropionic acid was as potent as a non-substituted fatty acid. The sulfoxide-homolog of tetradecylthioacetic acid, which is not activated to the CoA ester, did not activate PPAR. A coordinated mechanism would be consistent with the hypothesis that the hepatic changes triggered by peroxisome proliferators are a response to a general disturbance in fatty acid metabolism. Recent results (unpublished data) support the hypothesis that the ultimate PPAR-activating moiety is formed distal to fatty acid esterification with CoA and proximal to the oxidation of the acyl-CoA ester. It has been reported that induction of P4504A1 and fatty acyl-CoA oxidase by peroxisome proliferators are partly, if not entirely, due to an increase in the transcription rate of the respective genes (Reddy et al., 1986; Hardwick et al., 1987; Nemali et al., 1988). On the other hand, it is well known that high-fat diets can induce proliferation of peroxisomes accompanied by an increase in the activities of peroxisomal B-oxidation and fatty acid co-hydroxylation (Nilsson et al., 1984; Flatmark et al., 1988; Neat et al., 1980). Pure eicosapentaenoic acid, which is similar to the tetradecylthioacetic acid in that it is poorly oxidized by mitochondrial fl-oxidation, is reported to stimulate acyl-CoA oxidase activity (Aarsland et al., 1990a,b). Recently, Horie and Suga (1989, 1990) found that the increase in the quantity of acyl-CoA oxidase induced by high fat feeding was due predominantly to the decrease in the degradation rate of the enzyme rather than an increase in the synthetic rate. Figure 8 shows that at 24 hr after administration of 3-thiadicarboxylic acid, the acyl-CoA oxidase and P4504A1 mRNA levels are almost reduced to the control level, in contrast to the enzyme activities that increased up to 24 hr (Figs 9 and 10). Thus, at least two different mechanisms may be involved in the increase of hepatic acyl-CoA oxidase and P4504AI enzyme activities induced by poorly fl-oxidizable substrates (3-thia fatty acids). Whether the increase in the activity of the two enzymes after administration of 3-thia fatty acids is due somehow to a decrease in the rate of degradation or an increase in the rate of synthesis still remains to be evaluated. Since one possibility is that the receptor ligand is a fatty acid or fatty acid metabolite, the consequence of these results is that fatty acids may contribute to the induction of their own degradation and could represent a physiological counterpart to man-made, exogenous compounds that induce peroxisome proliferation. 6.2. THE CHEMICAL NATURE OF THE 3-THIA FATTY ACIDS AND METABOLIC REQUIREMENTSFOR ACTIVATORSOF THE P P A R

Chimaeric receptors encompassing the putative ligand-binding domain of PPAR are activated by peroxisomal proliferators and physiologically occurring concentrations of fatty acids, although these compounds are not thought to bind PPAR themselves (G6ttlicher et al., 1992). To be able to use the chimaeric receptor/reporter gene system in CHO cells for identifying potential ligands to PPAR, we tested whether the activation of the reporter gene in the cell model correlates with the induction of peroxisomal proliferation in vivo. The order of potency of peroxisomal proliferation-inducing drugs in vivo is reflected in the concentrations required for activation of the GR-PPAR chimaera (G6ttlicher et al., 1993; Issemann and Green, 1990). Furthermore, the weak peroxisomal proliferation inducing potential of co-3 polyunsaturated fatty acids in vivo corresponds to their higher PPAR-activating potency in vitro compared with C20:4 or saturated fatty acids, which is indicated by the lower concentrations required for receptor activation. Although the mechanism of PPAR activation is not yet clear, the dose-response relationship for activation of the chimeric receptor appears to reflect some of the characteristics that provide the basis for differential sensitivity to peroxisomal proliferators in vivo.

370

R.K. BERGEand E. HVATTUM

Thus, this cell model appears to be suitable for the analysis of the role of fatty acid metabolizing pathways in the activation of PPAR. In this way, it might be possible to identify potential ligands to PPAR that then can be tested for direct binding to the receptor in vitro. The nature of the transcription factors involved in the regulation of peroxisomal fl-oxidation genes is not fully elucidated. The peroxisome proliferator-binding protein isolated by Lalwani et al. (1987) appears not to be involved in the activation of genes required for peroxisome proliferation (Milton et al., 1988). The PPAR cloned by Isseman and Green (1990) and Grttlicher et al. (1992) could be activated by different peroxisome proliferators. However, the authors did not rule out the possibility that peroxisome proliferators may interact indirectly with the receptor. Finally, Hertz et al. (1991) have presented evidence that amphiphilic carboxylic peroxisomal proliferators may act as transcriptional activators of the thyroid hormone-dependent genes. Sulfur-substitution of the fl-carbon atom (Berge et al., 1989a) or perfluorination (Intrasuksri and Feller, 1991) blocks the accessibility of fatty acids to fl-oxidation, which presumably provides the molecular basis of their peroxisomal proliferation-inducing potential (Berge et al., 1989a,b). The higher potency of 3-thia-substituted comparted with the 4-thia-substituted fatty acids is reflected in the activation of the GR-PPAR chimaera in that the non-fl-oxidizable tetradecylthioacetic acid activates the receptor chimaera at a potency comparable to the strong peroxisomal proliferator WY14,643, whereas tetradecylthiopropionic acid is only as potent as a non-sulfur-substituted fatty acid. The lower activity of octathioacetic acid compared with that of tetradecylthioacetic acid might indicate the requirement for a minimal hydrophobic backbone. This proposition is consistent with the finding that shortening of the chain length to 6 carbon atoms abolishes the PPAR-activating potential (Grttlicher et al., 1993). An increased PPAR-activating potency is also discernible when comparing sulfur-substituted, non-fl-oxidizable dicarboxylic acids, with non-substituted dicarboxylic acids. For instance, 3-thiadicarboxylic acid is more potent than 1.16-hexadecanedioic acid. The in vitro data, however, do not show the higher potency of the sulfur-substituted dicarboxylic over the monocarboxylic acids in vivo. This may be dependent on differences in cellular uptake or in the pharmocokinetics in vivo between the groups of compounds. However, the toxicity of 1.16-hexadecanedioic acid at concentrations above 250~M indicates that the lack of receptor activation is not simply attributable to poor uptake into the CHO cells. Since the substitution of the fl-carbon atom does not interfere with the activation of a fatty acid to its ester with CoA (Aarsland and Berge, 1991), but blocks the subsequent step in the fatty acid degradation pathway (i.e. the unsaturation by acyl-CoA oxidase), it is likely that the acyl-CoA esters will accumulate in the cell and might constitute a key metabolite for the activation of PPAR. This hypothesis gains further support from the finding that a hydrophobic molecule carrying an acidic group, which is accessible to esterification with CoA, appears as the only common denominator amongst peroxisome proliferation-inducing compounds, including clofibric acid. Furthermore, inhibition of degradation of the CoA esters via the fl-oxidation pathway apparently enhances their potency as peroxisome proliferators (Lygre et al., 1986; reviewed in Lock et al., 1989). Peroxisome-proliferating xenobiotics represent a group of structurally diverse chemicals. The only common characteristics shared by these compounds appear to be their hydrophobicity and the frequent presence of a carboxylic acid functional group or a group that can be readily oxidized to such (Bronfman et al., 1986; Hawkins et al., 1987; Hertz et al., 1985; Lock et al., 1989). Compounds containing a carboxylic group are known to follow metabolic pathways in which the CoA thioester of the xenobiotic acid in question is an obligatory intermediate (Caldwell, 1984). Thus, it should not be surprising that carboxyl-containing peroxisome proliferating compounds are activated to their respective CoA thioesters (Fig. 14). Further suport for a key role of the acyl-CoA ester for PPAR activation comes from the observation that the sulfoxide-analog of tetradecylthioacetic acid does not induce the reporter gene, although it is also toxic at concentrations of 250/~M. The tetradecylsulfoxyacetic acid cannot be esterified with coenzyme A, at least under in vitro conditions (Aarsland and Berge, 1991). Thus, the lack of PPAR-activating and peroxisome-proliferating potential might be attributable to the lack of activation to the ester with CoA. The role of other fatty acid-metabolizing pathways besides fl-oxidation was assessed by using enzyme inhibitors or radical scavengers. The lack of any effect of indomethacin on the activation of the GR-PPAR chimaera, together with the finding that even saturated fatty acids can activate

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the chimeric receptor (G6ttlicher et al., 1992), suggests that the cyclooxygenase pathway does not play a major role in the formation of the ultimate PPAR-activating species. Reactive oxygen might play a role in PPAR activation similar to its role in the activation of the NF-x B transcription factor (Schreck et al., 1991). Antioxidants and radical scavengers, such as N-acetylcysteine and pyrrolidinedithiocarbamate (Schreck et al., 1991), however, do not substantially affect the activation of the GR-PPAR chimera. Furthermore, direct application of H202 does not activate the reporter gene, so that a causal link between reactive oxygen species and the activation of PPAR appears unlikely. The cytochrome P450 inhibitor SKF 525A reduces by 45% the induction of the reporter gene by C20:4. This might be taken as an indication of a role of cytochrome P450-dependent metabolism in the formation of the ultimate PPAR-activating molecule, which is consistent with the finding that the induction of cytochrome P4504AI is a primary event in the peroxisome-proliferator response (Bell and Elcombe, 1991). The data presented here, however, are not conclusive, since the other tested cytochrome P450 inhibitor, metyrapone, does not affect the induction of the reporter gene. Furthermore, if oxidation products of the fatty acids are the PPAR-activating species, then ~-hydroxylation and the subsequent dehydrogenation to the dicarboxylic acid would represent the obvious pathway. The lower activity of 3-thiadicarboxylic acid vs monocarboxylic acids in CHO cells, however, does not support this hypothesis. Peroxisome-proliferating compounds that cannot be activated to a CoA derivative have been described. Perfluorinated octane sulphonic acid administered to rats for 14 days induced peroxisome proliferation (Ikeda et al., 1987). Compared with other compounds, peroxisome proliferation caused by this potent, inducer is a relatively delayed response (10 days after a single intraperitoneal

372

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injection) (Haugbom and Spydevold, 1991). Thus, the question arises as to whether peroxisome proliferation with this compound is a secondary phenomenon due to its high toxicity or a direct effect of the compound per se. Also, leukotriene antagonists (tetrazole-substituted acetophenones) (Foxworthy et al., 1990) and dehydroepiandrosterone (Wu et al., 1989; Yamada et al., 1991) are reported to cause a remarkable increase in the number of peroxisomes and peroxisomal fl-oxidation. It has been previously postulated that the induction of peroxisome proliferation by various hypolipidemic drugs carrying a carboxylic function, as well as by metabolic stress, inducing fatty acids, is exerted through an increased cellular level of poorly or non-fl-oxidizable acyl-CoA esters (xenobiotic acyl-CoA thioesters) (Caldwell, 1984; Berge et al., 1987a,b). Thus, it has been suggested that the increase in the peroxisomal enzyme system in response to 3-thia fatty acid treatment may occur by a substrate-induced mechanism, i.e. by high cellular levels of long-chain acyl-CoA esters that are slowly oxidized by mitochondria (Bremer and Norum, 1982; Nilsson et al., 1984; Berge et al., 1987a,b, 1989b). Flatmark and co-workers (Flatmark et al., 1988; Fukami et al., 1986) have presented evidence that during high-fat feeding and clofibrate administration, gene expression is activated by components different from long-chain acyl-CoA. However, the authors did not rule out the possibility that the threshold value for the individual acyl-CoA is not changed. The level of long-chain acyl-CoAs increased rapidly in rat liver after tetradecylthioacetic acid feeding (Asiedu et al., 1990), and the 3-thia fatty acid can be activated to its CoA-thio-esters in vitro (Aarsland et al., 1990; Aarsland and Berge, 1991). Previously, it was found that clofibric acid can be activated in vivo (Lygre et al., 1986), which was confirmed by Bronfman et al. (1986, 1989). Thus, it seems well documented that many peroxisome proliferating compounds are activted to their respective CoA thioesters. This raises the possibility that it is not merely the amount of long-chain acyl-CoA, but also the nature of the acyl-CoA esters that are determinants for peroxisome proliferation. In this context, the 3-thia fatty acids, which are poorly oxidized by mitochondria, are of particular interest (Fig. 14). In summary, our present results are consistent with the idea that the ultimate PPAR activator is formed distal to the esterification of the inducing agent with CoA and proximal to its oxidation. It is tempting to speculate that acyl-CoA esters activate PPAR, but it remains to be clarified whether CoA-esters are themselves ligands to PPAR, or whether they serve indirect functions requiring further metabolism or secondary signalling pathways. If acyl-CoA esters are considered PPAR ligands, then the receptor and other acyl-CoA-binding proteins (Rasmussen et al., 1990) might interact in a similarly intricate way as retinoid-binding proteins and retinoid receptors do (Petkovich et al., 1987; Levin et al., 1992; Blomhoff et ak, 1990). Finally, the possible role of fatty acids in regulating PPAR activation and PPAR-mediated signal transduction might point out a more general role of newly discovered nuclear receptors in intermediary metabolite homeostasis.

7. EFFECTS ON CULTURED CELLS VS EXPERIMENTAL ANIMALS During the last few years, cell cultures have been frequently used to test, in a more direct manner, the effect of agents modulating peroxisomal biogenesis, P4504A1 and function in vivo (Gray et al., 1982, 1983). Considering the general problems of using primary cultures of hepatocytes and continuous cultures of different cell lines, caution should be taken in extrapolating findings from cell culture experiments to the in vivo situation. Thus, there are several reported differences between the in vivo and in vitro increase, e.g. in peroxisomal and P4504A1 activity. In vivo, the dicarboxylic derivative of tiadenol is as efficient as tiadenol itself (Berge et al., 1989a,b; Hvattum et al., 1991), while in vitro, this derivative showed no effect (Spydevold and Bremer, 1989). Moreover, in vivo, the tetradecylthiopropionic acid had small effects (Berge et al., 1989b), while in vitro this fl-oxidizable fatty acid analog is as efficient as tetradecylthioacetic acid in inducing peroxisomal enzymes (Spydevold and Bremer, 1989). When applied to studies on the effect of C22:1 fatty acids, it has been found that the cytotoxicity of the fatty acids prohibited high concentrations from being tested (Christiansen et al., 1985). In vivo, however, high fat diets and eicosapentaenoic acid induce the peroxisomal fl-oxidation enzyme system and peroxisome biogenesis (Christiansen et al., 1981;

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Kryvi et al., 1986; Aarsland et al., 1990b). In vivo diets high in ordinary fatty acids (monocarboxylic as well as dicarboxylic acids) result in almost no induction of peroxisomal//-oxidation enzymes (Thomassen et al., 1982; Berge et al., 1989a,b) while in cultured hepatoma cells, addition of such fatty acids stimulates peroxisomal fl-oxidation (Spydevold and Bremer, 1989). In the same cells, tetradecylthioacetic acid did not induce og-hydroxylation activity when the acyl-CoA oxidase activity was induced (unpublished data). This is in contrast to the in vivo situation, where tetradecylthioacetic acid induces both co-hydroxylation and acyl-CoA oxidase activity (Hvattum et al., 1991). Thus, using primary cultures of hepatocytes and continuous cultures, caution should be taken in extrapolating findings from cell culture experiments to the in vivo situation. Interestingly, in cultured Morris 7800 C1 hepatoma cells, tetradecylthioacetic acid reduced the acyl-CoA oxidase mRNA degradation rate 2-fold (Sorensen et al., 1993). The increase in the acyl-CoA oxidase mRNA steady state, and subsequently the enzyme activity by tetradecylthioacetic acid, therefore, is partly due to a prolonged half-life of the transcript in addition to an increased transcription rate. This has not been verified in vivo.

8. CARCINOGENICITY OF SULFUR-SUBSTITUTED FATTY ACID ANALOGS 8.1. CELLTRANSFORMATION Following the observation that many of the chemicals that induce hepatic peroxisomes are also hepatocarcinogenic, Reddy and Lalwani (1983) hypothesized a causal relationship between induction of the H202-producing enzyme fatty ACO and hepatocarcinogenesis. Despite data supporting an association between ACO activity and hepatocarcinogenesis, a causal relationship has not been established. Comparing peroxisome proliferators with widely different capacities to induce ACO and the carcinogenic potential of these proliferators could help to define the relationship between induction of ACO and subsequent formation of hepatic tumors. A possible correlation between induction of ACO activity and carcinogenic potential should be examined with different peroxisome proliferators using the same experimental protocol. If a strong inducer of peroxisomes failed to induce carcinogenesis, this would argue against a causal relationship between peroxisomal proliferation and the carcinogenic process. Several previous studies have addressed the possible quantitative relationship between the degree of peroxisome proliferation and hepatocarcinogenesis. Reddy et aL (1986) and Thomaszewski et al. (1986) compared peroxisome proliferation induced by several peroxisome proliferators to historical hepatocarcinogenicity bioassay data. Both groups concluded that the degree of peroxisome proliferation was correlated to the incidence of hepatocellular cancer. However, there are several potential problems in these comparative studies. Some of the doses of peroxisome proliferators used by Reddy et al. (1986) were not the same as those used in the carcinogenicity assays. In the studies performed by Thomaszewski et al. (1986), the animals were dosed by gavage, making it difficult to compare peroxisome proliferation quantitatively with the tumor incidence in previous bioassays where the chemicals were mixed in the diet. Finally, the evaluation of only one, single early time point in both studies makes it difficult to determine what role peroxisome proliferation may have on the progression of hepatic tumors. Cell transformation assays appear to measure at least some of the stages in the progression of a cell from the normal to the malignant state. The C3H10T1/2 line of mouse embryo fibroblasts (10T1/2 cells) used in cell transformation assay is particularly appealing in that it measures both initiating and promoting events. We already have published that the hypolipidemic drug clofibrate has a tumor-promoting potential, but no direct transforming activity in vitro using 101/2 cells (Lillehaug et al., 1986). In the same work, the hypolipidemic and potent peroxisome-proliferating drug tiadenol had no tumor-promoting potential. In our hands, the ACO activity is readily measurable in the 101/2 cells, in spite of a low basal activity. The order of potency with respect to induction of ACO activity in this cell line was tetradecylthioacetic acid > 3-thiadicarboxylic acid > clofibric acid > tetradecylthiopropionic acid (Aarsaether, 1990). This order was different in most aspects from the in vivo situation (Berge et aL, 1989a). In vivo, there was considerable breakdown of the drugs and

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subsequent metabolites (Figs 2, 3 and 4). The spectrum of metabolites in culture may not reflect the in vivo situation and could influence the carcinogenic and/or tumor-promotion potential. Our results showed that neither 3-thia dicarboxylic acid nor tetradecyl thioacetic acid and tetradecyl thiopropionic acid were active as carcinogens in a transforming test system (unpublished data). The sulfur-substituted fatty acid analogs showed variable degrees of tumor-promoting capacity, but treatment of cells with all of these analogs resulted in enhanced morphological transformation (Aarsaether, 1990). However, the number of morphologically transformed foci (3-5) was much lower than that seen in the clofibrate group (8 type-III foci) and higher than that in the control group (1-3). Thus, the order of potency with respect to tumor promotion in the cell line was clofibric acid > tetradecylthioacetic acid > 3-thiadicarboxylic acid = tetradecylthiopropionic acid, making the relationship between certain peroxisomal enzymes and tumor promotion in the C3H/10T1/2 C1 8 cells doubtful (Aarsaether, 1990). Our results are consistent with other laboratories using cell transformation assays to elucidate possible mechanisms of hepatocarcinogenesis by peroxisome proliferations. Sanchez et al. (1987) have shown that the plasticizer di-(-2ethylhexyl)phthalatas (DEHP), and its metabolite mono(2-ethylhexyl)phthalata did not produce oncogenic transformation, initiate the process of transformation nor promote the process of transformation in cultures pretreated with a chemical carcinogen. Mikalsen et al. (1990) have used the Syrian hamster embryo cell transformation assay, which is sensitive not only to chemicals that affect the primary DNA sequence, but also to agents that cause aneuploidy. In their hands, there was no correlation between transforming activity and induction of peroxisomal enzymes. In view of the results obtained in different laboratories, the transforming potential of peroxisome proliferators was not dose dependent and did not correlate with an increase in peroxisomal volume and associated enzyme activities, implying a minor role of peroxisomes in the process of transformation by these xenobiotics. The cellular lipid composition of C3H/10T1/2 cells can be effectively modified by adding fatty acids into the media, without perturbing cellular integrity and morphology. The fatty acids themselves also affect the promotion stage of carcinogenesis, probably by changing the membrane lipid fluidity. It is well known that the peroxisome proliferators, to different degrees, affect fatty acid metabolism. Whether the peroxisome prolfierators used in thsi study affect the membrane lipid fluidity should be considered. 8.2. HEPATOCARCINOGENESIS

C3H mice are susceptible to chemically-induced hepatocarcinogenesis, and a promoting effect of peroxisome proliferators in this strain already has been seen. In the mouse strain C3H/Hej, 3-thiadicarboxylic acid produced intensive hepatomegaly and induction of ACO, whereas tetradecylthioacetic acid and clofibrate affected these parameters to a lesser extent. The steady state of liver enlargement and increase in peroxisomal enzymes were reached with 14 days after starting the administration of peroxisome proliferators. Thus, the levels of these parameters are nearly constant during long-term feeding experiments (Aarsaether, 1990). The carcinogenecity of the above fatty acid analog was compared in the C3H/Hej mouse strain. Before the mice were fed a diet containing sulfur-substituted fatty acid analogs, one or two groups of mice had been given a single i.p. injection of diethylnitrosamine (0.5 #mol/g body weight), hereafter referred to as the 'initiated group'. With respect to this group, the experiments were terminated after 3 and 6 months, whereas in the experiments with the 'non-initiated group' (in the absence of diethylnitrosamine), no visible tumors were observed after feeding a diet containing only the sulfur-substituted fatty acid analog for 12 months. Moreover, during this period, no animals died after 3-thia fatty acid administration, whereas two animals died in the control group. However, in the 'initiated' group, the sulfur-substituted fatty acid analogs increased the incidence of neoplastic foci after 3 months of feeding and enhanced the yield of tumors after 6 months of feeding (Aarsaether, 1990). In C3H/HeJ mice fed sulfur-substituted fatty acid analogs, a parallel between the induction of ACO and hepatic tumor promotion is lacking (Aarsaether, 1990). 3-Thiadicarboxylic acid feeding does not induce tumor promotion in liver at a dosage that markedly affects liver physiology,

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including peroxisome proliferation. Tetradecylthioacetic acid, which induces peroxisomal proliferation to less than half the level obtained by 3-thiadicarboxylic acid, has a low tumor-promoting potential (Aarsaether, 1990). If the oxidative stress hypothesis is correct, there should be some relationship between the magnitude of peroxisome proliferation and subsequent tumor formation. Certainly, the more potent peroxisome proliferators, such as ciprofibrate and Wy-14,643, produce a high incidence of liver tumors at much lower doses than weaker compounds, such as DEHP (Fahl et al., 1984; Marsman et al., 1988). However, in studies where H202 steady state levels were determined, a correlation with carcinogenic potency was not obtained (Thomaszewski et al., 1986). Comparative studies between Wy-14,643 and DEHP failed to correlate differences in liver tumor formation with differences in peroxisomal proliferation and effects on H202-producing and -metabolizing enzymes (Marsman et al., 1988; Conway et al., 1989). Likewise, Mochizuki et al. (1982) have shown that there are no dose-related increases in liver tumors in rats after administration of clofibrate at different concentrations. Although the literature contains evidence that peroxisome-proliferator-induced hepatocarcinogenicity is associated with oxidative stress, definitive evidence that this is the primary mechanism responsible for tumor formation is lacking. An alternative suggestion might be to look for perturbations at the membrane level as a possible mechanism for the development of hepatic tumors after administration of peroxisome proliferators. Peroxisome proliferators are compounds that contain the carboxylic acid group (-COOH), or they can readily be converted into such compounds. In recent years, attention has been drawn to the toxicity of a variety of carboxylic acids and their derivatives. It is known that many xenobiotics with carboxyl groups from CoA-esters in vivo. It has been suggested by Bronfman et al. (1986, 1989) and by our laboratory (Berge et al., 1989a,b; Aarsland et al., 1989) that the pharmacologically active species of the peroxisome proliferators may be their acyl-CoA thioesters. The normal functioning of living cells depends, to a large extent, upon the properties of various membranes in the cells. Lipid-related xenobiotics, notably hybrid triglycerides and cholesterol esters, have the potential to enter membranes and alter membrane structure. This, in turn, could modify membrane-dependent processes, either by structural alteration or by slowing the deacylation-reacylation of fatty acid residues within the membrane. This could finally activate enzymes like phosphokinase C, thus altering cell proliferation. To illustrate the potential complexity of xenobiotic-lipid relationships, the role of diacylglycerols in cell signalling is of potential importance. The tumor-promoting phorbol esters have been postulated to mimic the action of diacylglycerols, which transiently activate protein kinase C (Nishizuka, 1984). This enzyme appears to play a crucial role in signal transduction between the cells and their environments. Xenobiotic diacylglycerols could behave similarly as substitutes for the natural analogs, producing a permanently activated enzyme.

9. CONCLUDING REMARKS AND FUTURE ASPECTS The possible mechanisms of peroxisome proliferation have been discussed. The receptor hypothesis and the substrate overload-perturbation of lipid metabolism theory are not mutually exclusive. Specific lipid accumulation may be a key event in peroxisome proliferation, although inhibition of mitochondrial fatty acid oxidation is not necessarily an essential requirement. Other mechanisms may be operative. It is conceivable that certain peroxisome-proliferating chemicals may coincidentally recognize an endogenous receptor or other regulatory sites involved in the regulation of lipid metabolism. Alternatively, the xenobiotics may induce a putative endogenous ligand for a given receptor (i.e. free fatty acids or their CoA derivatives). In any of these instances, the xenobiotics or the putative endogenous ligands indirectly may activate the receptor or a regulatory enzyme. Several key enzymes in lipid metabolism are known to be regulated by protein kinases (Hardie, 1989). Protein kinase C activity is also known to be regulated by the lipid environment. This occurs either directly by diglycerides or more indirectly by the surrounding membrane phospholipids. At both levels, alkyl substitution is known to alter enzyme function. Recently, Orellana et al. (1990)

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have shown that both naturally occurring acyl-CoAs and acyl-CoA thiesters of several peroxisome proliferators strongly increase the activity of rat liver protein kinase C. Although still speculative, it is tempting to propose a common regulatory step (e.g. the protein kinase C system) to account for peroxisome proliferation induced by both chemicals and pathophysiological-metabolic states (high fat diets, starvation and diabetes). This inductive response would not be strictly related to either the quantity of xenobiotoc-CoA or endogenous long-chain acyl-CoA alone, but rather to the qualitative and quantitative nature of the hepatic acyl-CoA content. Some peroxisome-proliferating regimes may act primarily at a pre-hepatic level (high-fat diets, starvation and diabetes) and subsequently alter the hepatic metabolism of fatty acids (i.e. enhance hepatic influx of fatty acids). Alternatively, the inducing agent (peroxisome-proliferating compounds) may act directly within the liver, perturbing lipid metabolism. In either instance, the altered hepatic CoA pool is suggested to be the proximate inducer. To what extent does peroxisome proliferation have any relevance for humans? There is a great difference in responsiveness among different species to proliferating agents (Watanabe et al., 1989). Peroxisome proliferation in humans during treatment with hypolipidemic agents known to be peroxisome proliferating in rodents is claimed by some investigators (Hanefeld et al., 1982), while others have questioned it (Cariot et al., 1986, 1987). A number of compounds have been associated with an increased number of peroxisomes in humans (Phillips et al., 1987), probably as an idiosyncratic reaction. The most consistent reports about human peroxisome proliferation are observations made during acute perturbations of mitochondrial fatty acid oxidation (acyl-CoA dehydrogenase defects, Reyes syndrome) (Partin et al., 1971; Treem et al., 1986). Actually, peroxisomal fl-oxidation induced in these instances has been suggested to function as a salvage pathway against the accumulation of unfavourable levels of fatty acid metabolites. The major significance of induced peroxisomal proliferation in rodents may be its validity as an experimental model to penetrate further the essential role peroxisomes play in mammalian metabolism (Mannaerts and Van Veldhoven, 1990; Moser, 1989), and substituted fatty acid analogs are useful tools for this purpose. The desirable lipid-lowering effect of substituted and polyunsaturated fatty acids deserves further attention. Further research may extent our knowledge regarding the interplay between hepatic lipid metabolism and serum lipids. As a therapeutic principle, the lipid-lowering effect of the substituted fatty acids awaits additional study. Aside from their hypolipidemic effect, the most promising therapeutic potential of the nonmetabolizable long-chain fatty acids may relate to their antiobesity effect and the hypoglycemic-hypoinsulinemic effects in hyperlipidemic-obesity
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