Narendra D. Lalwani

Evaluation of Selected Hypolipidemic Agents for the Induction of Peroxisomal Enzymes and Peroxisome Proliferation in the Rat Liver

There is a considerable interest in developing potent and safe hypolipidemic drugs for the prevention and management of coronary heart disease in man. In rodents, many of these hypolipidemic compounds induce hepatomegaly, proliferation of peroxisomes and a polypeptide with an approximate mol. wt. of 80000 in liver cells. In the present study, we have examined 10 hypolipidemic compounds for the induction of peroxisome proliferation associated 80000 mol. wt. polypeptide (polypeptide PPA-80), peroxisomal enzymes and peroxisome proliferation in rat liver, in view of the emerging evidence that hepatic peroxisome proliferators as a class are carcinogenic in rats and mice. All ten compounds, fenofibrate (isopropyl-[4-( p-chlorobenzoyl)2-phenoxy-2-methyl] propionate; LS 2265 (taurine derivative of fenofibrate); bezafibrate (2-{4-(2-[4-chlorobenzamido)ethyl] phenoxy}-methyl propionic acid; gemfibrozil (5-2[2,5-dimethylphenoxy]2-2-dimethylpentanoic acid); methyl clofenapate (methyl-2-[4-(p-chlorophenyl)phenoxy]-2-methyl propionate); DG 5685 (5-[4-phenoxybenzyl]trans-2-(3-pyridyl)1,3-dioxane), DH6463 (5-[4-phenoxybenzyl] trans-2-(3-pyrimidinyl)-1,3-dioxane); tiadenol(bis[hydroxyethylthio]-7, 10-decane); ciprofibrate (2,-[4-{2,2-dichlorocyclopropyl}-phenoxy]2-methyl propionic acid) and RMI-14,514 ([5-tetradecycloxyl-2-furancarboxylic acid), produced a marked but variable increase in the activities of peroxisomal enzymes catalase, carnitine acetyltransferase, heat-labile enoyl-CoA hydratase and the fatty acid β-oxidation system and in the amount of polypeptide PPA-80 as demonstrated by SDS-polyacrylamide gel electrophoresis. The peptide map patterns of polypeptide PPA-80 in liver induced by these compounds were strikingly similar. The ultrastructural studies demonstrate that fenofibrate, ciprofibrate, LS 2265, DG 5685 and DH 6463 can induce proliferation of peroxisomes in liver cells of rats, and further confirm the previous reports of hepatic peroxisome proliferative activity of methyl clofenapate, tiadenol, bezafibrate, gemfibrozil and RMI-14514, as shown morphologically. Whether these structurally unrelated chemicals or their metabolite(s) directly activate the peroxisome specific genes to induce this multi-enzyme system or they exert their action on peroxisomes indirectly by causing fatty acid overload in hepatocytes remains to be elucidated. These chemicals offer a simple and reproducible means of stimulating peroxisomal enzymes in liver and should serve as useful tools, for evaluating the implications of hepatic peroxisome proliferation and in elucidating the mechanism of peroxisome proliferator-induced carcinogenesis.

Detection of a nafenopin-binding protein in rat liver cytosol associated with the induction of peroxisome proliferation by hypolipidemic compounds

[3H]nafenopin, a known inducer of liver peroxisomal enzymes, was shown to bind to a specific, saturable pool of binding sites in cytosols from rat liver and kidney cortex. Tissue levels of this binding protein (liver greater than kidney cortex; not detectable in myocardium, skeletal muscle) were seen to correlate with the ability of nafenopin to induce peroxisomal enzymes in these organs. Clofibrate and ciprofibrate, which are structurally similar to nafenopin, competitively blocked the specific binding of [3H]nafenopin. Phenobarbital, a non-inducer of peroxisomes, and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid and 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)acetamide, which are structurally unrelated peroxisome proliferators, did not complete for the specific [3H]nafenopin binding sites. The [3H]nafenopin binding protein is proposed as a mediator of the drug-induced increase in peroxisomes and associated peroxisomal enzymes.

Chemical Carcinogens Without Mutagenic Activity: Peroxisome Proliferators As A Prototype*

Chemical carcinogens can be classified into two categories (i.e. mutagenic and non-mutagenic) on the basis of positive or negative evidence of DNA damage, mutagenicity or chromosomal aberrations in short-term test systems. Evidence indicates that carcinogenic peroxisome proliferators are negative in short-term test systems. This paper outlines approaches which may be useful in identifying a chemical carcinogen without mutagenic activity. It is conceivable that an alteration in DNA, if essential for initiation of neoplasia, may be mediated indirectly by the biological effects of nonmutagenic carcinogens.

Induction of fatty acid β-oxidation and peroxisome proliferation in the liver of rhesus monkeys by DL-040, a new hypolipidemic agent

Many structurally unrelated hypolipidemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in liver parenchymal cells of rodents, but there is relatively limited evidence regarding the ability of such compounds to induce peroxisome proliferation in the livers of nonrodent species including man. The present study was designed to determine if DL-040 (4-(((1,3-benzodioxol)-5-yl)methyl)amino-benzoic acid), a newly developed hypolipidemic agent, induces peroxisome proliferation in the liver of adult rhesus monkeys. Feeding of DL-040 (300 mg/kg body wt for 1 week; and 400 mg/kg body wt for 10 weeks) caused a significant increase in peroxisome population as determined by ultrastructural and morphometric analyses. The DL-040-induced peroxisome proliferation was accompanied by increases in the levels of catalase, carnitine acetyltransferase and the peroxisomal fatty acid beta-oxidation system. As expected, DL-040 caused a significant reduction of serum cholesterol and low density lipoprotein content. These data suggest that hepatic peroxisome proliferation is inducible in nonhuman primates at dose levels that exceed therapeutic levels.

Lack of covalent binding of peroxisome proliferators nafenopin and WY-14 643 to DNA in vivo and in vitro

[3H][2-methyl-2-p-(1,2,3,4-tetrahydro-naphthyl)phenoxy] propionic acid (nafenopin), a hepatocarcinogenic peroxisome proliferator, when administered p.o. to normal intact and partially hepatectomized male F344 rats did not show any significant binding to DNA and RNA, but bound to proteins. The in vitro incubation of [3H]nafenopin and [3H]4-chloro-[6-(2,3-xylidino)pyrimidinylthio]acetic acid (Wy-14643), another peroxisome proliferator, with hepatic microsomes and calf thymus DNA also showed no significant binding of these chemicals to DNA.

Induction of Hamster Hepatic Peroxisomal β-oxidation and Peroxisome Proliferation-associated 80000 mol. wt. Polypeptide by Hypolipidemic Drugs

1 The effects of hypolipidemic drugs fenofibrate (isopropyl[4-(p-chlorobenzoyl)-2-phenoxy-2-methyl]propionate), pyrinixil (BR-931; [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-β-hydroxyethyl) acetamide]), methyl clofenapate (2-methyl-2[4-(p-chlorophenyl)phenoxy]2-methyl-propionate), and clofibrate (ethyl α-p-chloro-phenoxyisobutyrate) on plasma triglyceride levels, hepatic peroxisome proliferation and peroxisome-associated enzymes in hamsters were investigated. 2 Fenofibrate, pyrinixil and methyl clofenapate were administered in the diet at 0.2% level (w/w) for 6 weeks. Clofibrate was fed at 0.5% level. 3 Fenofibrate, pyrinixil and methyl clofenapate induced a marked proliferation of peroxisomes in hamster liver cells which was comparable to that observed in the rat and mouse liver, whereas the peroxisome proliferative effect of clofibrate was less pronounced. Peroxisomal fatty acid β-oxidation system was found in the hamster liver and its activity was enhanced significantly by hypolipidemic drugs. The magnitude of induction of [1-14C]palmitoyl CoA oxidation, heat-labile enoyl-CoA hydratase and peroxisome proliferation-associated 80000 mol. wt. polypeptide in the hamster appeared to parallel the extent of peroxisome proliferation. 4 All four hypolipidemic compounds increased hepatic catalase and carnitine acetyltransferase activities and decreased plasma triglyceride levels in the hamster. The observed hepatic effects of hypolipidemic drugs in hamster are identical to those induced by peroxisome proliferators in the rat and mouse. 5 These observations suggest that hypolipidemic agents identified as peroxisome proliferators in rats, mice and now hamsters would very likely enhance the peroxisomal enzyme system in other species. Additional information on the interspecies responses to peroxisome proliferators, however, is necessary to assess the role of peroxisome proliferation in carcinogenesis.