Noninhibitory Effect of Antioxidants Ethoxyquin, 2(3)

Home , Butylated hydroxytoluene

[CANCER RESEARCH 43, 1680-1687, April 1983] 0008-5472/83/0043-OOOOS02.00 Noninhibitory Effect of Antioxidants Ethoxyquin, 2(3)-7ert-butyl-4- hydroxyanisole and 3,5-Di-tert-butyl-4-hydroxytoluene on Hepatic Peroxisome Proliferation and Peroxisomal Fatty Acid /^-Oxidation Induced by a Hypolipidemic Agent in Rats1

Narendra D. Lalwani, M. Kumudavalli Reddy, Saeed A. Qureshi, Charles M. Moehle, Hidenori Hayashi,2 and Janardan K. Reddy3

Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60617

ABSTRACT the hypothesis that peroxisome proliferator carcinogenesis is mediated by H2O2 and that antioxidants by inhibiting lipid per 6-Ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin) oxidation could retard or inhibit this process. and the phenolic antioxidants 2(3)-iert-butyl-4-hydroxyanisole and 3,5-di-fe/r-butyl-4-hydroxytoluene inhibit the development of tumors induced by several chemical carcinogens that are geno- INTRODUCTION toxic. The antioxidants appear to exert these protective effects The pioneering work of Wattenberg (60, 61 ) has established by inhibiting metabolic activation and by enhancing enzyme that the dietary administration of antioxidants such as systems that facilitate the detoxification of electrophilic form(s) ethoxyquin,4 BHA, and BHT can inhibit the induction of cancer of carcinogens. Information regarding the protective effects, if in a variety of rodent tissues by structurally diverse chemical any, of antioxidants against carcinogenesis by chemical carcin carcinogens including benzo(a)pyrene, 7,12-dimethylbenz(a)- ogens that do not appear to generate electrophilic or mutagenic anthracene, diethylnitrosamine, uracil mustard, and methylaz- metabolites is, however, lacking. To examine the proposal that oxymethanol acetate. The mechanism(s) by which these syn the carcinogenicity of such nonmutagenic compounds is due to thetic antioxidants inhibit chemical carcinogenesis has not been their ability to initiate lipid peroxidation chain reactions either fully elucidated, but recent investigations have demonstrated an directly or indirectly, we plan to systematically investigate the array of alterations in the activities of several carcinogen-detox effects of antioxidants on hepatocarcinogenesis by peroxisome ifying hepatic enzymes in rats and mice (4, 6, 16, 24, 28, 49, proliferators. We have studied the acute effects of antioxidants 50). Studies with BHA have shown that this antioxidant causes ethoxyquin, 2(3)-fe/t-butyl-4-hydroxyanisole, and 3,5-di-fert-bu- a reduction of overall metabolism, DMA binding, and mutagenic tyl-4-hydroxytoluene on the induction, in the rat liver, of hepatic activity of benzo(a)pyrene (4, 7). Therefore, it is envisaged that peroxisome proliferation and peroxisome-associated enzymes the protective effects of antioxidants are, in part, due to a shift by 2-[4-(2,2-dichlorocyclopropyl)phenoxy]-2-methyl propionic in the equilibrium between metabolic activation and detoxification acid (ciprofibrate), a potent peroxisome proliferator. Ethoxyquin of the carcinogen (7, 24, 61). Recently, Cha and Heine (7) at 0.5% (w/w) dietary level exerted no inhibitory effect on the proposed that the enhancement of these detoxification enzyme induction by ciprofibrate (0.1%, w/w) of hepatomegaly, peroxi activities is actually designed to accelerate the elimination of some proliferation (volume and numerical density), catalase, administered antioxidant, which inadvertantly confers protection carnitine acetyltransferase, heat-labile peroxisomal enoyl-coen- against the electrophilic species of chemical carcinogens (36). zyme A hydratase, and peroxisomal fatty acid 0-oxidation sys Although the antioxidant-induced alterations in the metabolism tem. Both 2(3)-fert-butyl-4-hydroxyanisole (0.7%, w/w) and 3,5- of carcinogens, leading to a shift in the production of electrophilic di-ferf-butyl-4-hydroxytoluene (0.7 or 0.07%) also failed to inhibit reactants, appear pertinent, the possibility that the inhibitory the ciprofibrate-induced hepatomegaly and hepatic peroxisome effect of antioxidants on cancer development is mediated proliferation. Additionally, none of these antioxidants altered the through a reduction of free radical reactions and inhibition of lipid ciprofibrate-induced increase in M, 80,000 peroxisome prolifera peroxidation remains to be elucidated. Damage to membranes, tion-associated polypeptide in liver as analyzed by sodium do- DMA, and other macromolecules by reactive oxygen intermedi decyl sulfate:polyacrylamide gel electrophoresis. The similar in ates and the lipid peroxidation chain reactions that follow has creases in peroxisome population and elevations of H202-pro- been suggested as a major cause of cancer and aging (17, 18, ducing peroxisomal fatty acid /3-oxidation system suggest that 31,56). This may be particularly relevant in the case of a growing excess levels of intracellular H202 are generated in the livers of list of chemical carcinogens such as hypolipidemic peroxisome rats fed ciprofibrate either alone or in combination with a dietary proliferators, pesticides, and other xenobiotics that have not antioxidant. These experiments provide a model system to test been shown to generate detectable mutagenic or DNA-damaging

1This work was supported by Grants CA-32504 and GM-23750. metabolites (2, 3, 57, 59). 2 Present address: Department of Physiological Chemistry, Josai University, 1- 1, Keyakidai, Sakado, Saitama, 350-02, Japan. 3 To whom requests for reprints should be addressed, at the Department of * The abbreviations and trivial names used are: ethoxyquin, 6-ethoxy-1,2-dihy- Pathology, Northwestern University Medical School, 303 East Chicago Avenue, dro-2,2,4-trimethylquinoline; BHA, 2(3)-tert-butyl-4-hydroxyanisole; BHT, 3,5-di- Chicago, III. 60611. Ãert-butyl-4-hydroxytoluene;ciprofibrate,2-(4-{2,2-dichlorocyclopropyl)phenoxy]-2- Received August 20, 1982; accepted December 30, 1982. methyl propionic acid; CoA, coenzyme A; SDS, sodium dodecyl sulfate.

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Studies in our laboratory have shown that the dietary admin administered in powdered chow at the dietary concentration of 0.1% (w/ istration of 6 structurally diverse hypolipidemic compounds, that w) for 6 weeks. The antioxidants were also administered in the diet for are capable of inducing profound proliferation of peroxisomes in 6 weeks at the levels indicated, alone or in combination with 0.1% liver parenchymal cells (42, 46, 48), leads to the development of ciprofibrate:BHT at 0.7 or 0.07%, BHA at 0.5%, or ethoxyquin at 0.5% (60). The control rats were fed powdered chow without the added hepatocellular carcinomas in rats and mice (41, 47). Recent chemicals. After the treatment, the animals were killed by cervical dislo reports, from other laboratories, of carcinogenicity of the hypo cation under light ether anesthesia, and their gross body and liver weights lipidemic drugs clofibrate (52), gemfibrozil (12), bezafibrate (19), fenofibrate,5 and ciprofibrate6 and of certain industrial plasticizers were determined. Subcellular Fractionation of Liver. The livers were homogenized in such as di-(2-ethylhexy)phthalate and di-(2-ethylhexyl)adipate ice-cold 0.25 M sucrose (10% homogenate, w/v), using a Potter-Elvehjem (10), all of which induce peroxisome proliferation (21, 45, 48), homogenizer. These homogenates were fractionated into nuclear (700 are consistent with the hypothesis that potent hepatic peroxi x g for 10 min), heavy mitochondrial (11,000 x g for 3 min), and light some proliferators as a class are carcinogenic (41). Accumulation mitochondrial (15,000 x g for 15 min) fractions in a Beckman J-21C centrifuge at 4Â°according to the procedure outlined by Baudhuin ef al. of abundant quantities of autofluorescent lipofuscin, an indicator of oxidative polymerization reactions involving lipids and proteins (5). The postlight mitochondrial supernatants were centrifugea at 105,000 x g for 1 hr using a Ti 50 rotor in a Beckman L5-65 ultracentri- (11,15, 31, 53), has been observed in liver during hepatocarcin- fuge to obtain the microsomal fraction and postmicrosomal soluble (cell ogenesis by peroxisome proliferators (44). Whether this pigment sap) proteins. All pellets were washed by resuspending in a 0.25 M accumulation in liver can be regarded as evidence for increased sucrose:0.1% ethanol solution and recentrifuged at the respective g H2O2 toxicity and lipid peroxidation reactions and by inference forces. for the initiation of neoplastic change by the metabolic by The large particle fraction (heavy and light mitochondrial fractions) of products of peroxisome proliferation is not certain. normal and ciprofibrate-treated rat livers was fractionated by the sucrose Because inhibition of lipid peroxidation has been proposed as density gradient centrifugation as described by Hayashi ef al. (20). one of the mechanisms responsible for the anticarcinogenic Enzyme Assays. The activities of several peroxisome-associated property of antioxidants (17, 18, 31, 60, 63), it appears worth enzymes such as catalase (33), carnitine acetyltransferase (14), heat- labile enoyl-CoA hydratase (39, 51), and the palmitoyl-CoA oxidizing while to investigate the modulating effects of antioxidants on system (30) were also measured. Protein concentrations were deter hepatocarcinogenesis by peroxisome proliferators. Available evi mined by the method of Lowry ef al. (32) using crystalline bovine serum dence indicates that the accumulation of lipofuscin occurring albumin as the standard. during the normal aging process can be slowed by free radical- SDS:Polyacrylamide Gel Electrophoresis. The postnuclear, large quenching antioxidants (53, 54, 63). As an initial step, we have particle, and microsomal fractions of liver were prepared (43) and ana investigated the early effects of simultaneous administration of lyzed by SDSipolyacrylamide gel electrophoresis according to the an antioxidant (ethoxyquin, BHA, or BHT) with a peroxisome method of Laemmli (25) to ascertain the effect of antioxidants on the proliferator on peroxisome proliferation and peroxisome-associ- induction by ciprofibrate of a M, 80,000 peroxisome proliferation-asso ated enzymes in rat liver. The results demonstrate that antioxi ciated polypeptide (43). dants do not prevent peroxisome proliferation and induction of Morphology and Morphometry. Small pieces of liver were processed peroxisome-associated enzymes in livers of rats fed ciprofibrate, for electron microscopy (38). One-^m-thick sections of Epon-embedded liver tissue were stained with toluidine blue and examined in a Zeiss a potent peroxisome proliferator (27). Since antioxidants fail to prevent peroxisome proliferation and induction of peroxisome- Ultraphot III microscope. Thin sections were examined in an electron microscope. associated enzymes by a hypolipidemic agent, long-term studies For morphometric analysis of changes in peroxisome volume density, with this model system will enable us to examine the hypothesis 30 randomly photographed electron micrographs of cytoplasm of liver that the anticancer effect of antioxidants is due to their free cells from each experimental group (10 electron micrographs from one radical-scavenging property. or 2 blocks/animal; 3 animals/group) were obtained. Micrographs were taken at x8,000 and enlarged 2.5 times at printing to a final magnification of X20.000. Points of intersection overlying cytoplasm, mitochondria, MATERIALS AND METHODS and peroxisomes were counted, using a 5-mm spaced lattic grid. The Chemicals. The hypolipidemic compound, ciprofibrate (white odorless volume density of mitochondria and peroxisomes was determined in powder; purity, 99.8%; M, 289.2), was a generous gift from Dr. H. P. relation to cytoplasmic volume according to the method described by Drobeck, Sterling-Winthrop Research Institute, Rensselaer, N. Y. The Weibel (62). antioxidants BHA and BHT were purchased from Sigma Chemical Co., St. Louis, Mo.; ethoxyquin was donated by Monsanto Industrial Chemi RESULTS cals Co., St. Louis, Mo. [1-14C]Palmitoyl-CoA (specific activity, 59 mCi/ mmol) was obtained from Amersham Corp., Arlington Heights, III. Cro- Hepatomegaly. As was observed previously (27), dietary tonyl-CoA, s-palmitoyl-CoA, NAD*, NADP+, flavin adenine dinucleotide, administration of the hypolipidemic compound ciprofibrate to rats CoA, and crystalline bovine serum albumin were obtained from Sigma produced marked enlargement of the liver. Administration of the Chemical Co. antioxidants ethoxyquin, BHA, or BHT to rats has also produced Treatment of Animals. Male F344 rats (100 to 150 g), obtained from significant enlargement of the liver but not to the same extent Charles River Breeding Laboratories, Wilmington, Mass., were housed as that caused by ciprofibrate. Concurrent dietary administration singly in hanging stainless steel wire cages, with a 12-hr light-dark photo of ciprofibrate with one of the antioxidants produced enlargement period. They had free access to chow and water. Ciprofibrate was of liver comparable to or slightly greater than that produced by ciprofibrate alone (Tables 1 and 2). For example, relative liver 5G. F. Blane (Laboratoires Furnier, S. A., Dijon, France), personal communica weights in rats fed ciprofibrate and ethoxyquin was 12.4%, tion. 6H. P. Drobeck (Sterling-Winthrop Research Institute, Rensselaer. N. Y.), per compared to 9.3% in rats fed only ciprofibrate and 6.7% in rats sonal communication. fed ethoxyquin alone.

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Table 1 Peroxisomeproliferation-associatedchanges in liver in male F344 rats fed an antioxidant ethoxyquinand hypolipidemic compound ciprofibrate Ciprofibrate and ethoxyquin were mixed in powdered chow, 0.1 and 0.5% levels, respectively. The enzyme activities were determined in liver homogenates as described in "Materials and Methods." hydratase (f

Table 2 1.0 2o Effect of antioxidants BHT and BHA on the induction of peroxisomal enzymes cafa/ase and the palmitoyl-CoA oxidation system in livers of male F344 rats ted ciprofibrate The antioxidants were mixed in diet, at concentrations indicated below, with or without 0.1% ciprofibrate. The animals were fed ad libitum for 6 weeks. In wt (g/ catalase Concentra 100 g body activity (units/ CoA oxidation (%)ControlsBHTBHACiprofibrate+BHT+BHA0.70.070.50.10.70.070.5Livertion wt)3.5 mgprotein)45414839941091187533259"9Â°10*13"[1-l4C]Palmitoyl-(jimol/min/gliver)1.23 +0.4a5.0 Â±0.221.52 1.0 Â±0.4Â°3.4 Â±0.241 1b 2b +0.54.3 .47 Â±0.701.38 Â±0.2C9.3 Â±0.117.2 OS Â±0.4"10.2 Â±0.55Â°15.36 +0.4o8.7 +0.33015.40 +1.3610.0 +0.42Â°17.5 + 0.46Liver + 0.30o a Mean Â±S.D.of 4 to 6 animals/group. 6 N M L P t M L r % 6 Significantlydifferent from controls (p < 0.001). * c Significantlydifferent from controls (p < 0.05). 2o 1.0r 3a K

Liver Peroxisomal Enzymes. The effects of dietary adminis tration of ethoxyquin and/or ciprofibrate on specific activities of peroxisome-associated enzymes in male rat liver are shown in Table 1. As expected, the liver homogenates prepared from ciprofibrate-treated rats demonstrated significantly higher activi ties of catalase, carnitine acetyltransferase, and palmitoyl-CoA 1.04bnt3b- oxidizing system when compared to liver homogenates obtained from either control or ethoxyquin-fed animals. The total enoyl- CoA hydratase activity in liver homogenates of rats fed ciprofi 0.8 brate was also increased; over 97% of this induced activity was found to be heat-labile peroxisomal enzyme (Table 1). This clearly indicates that ciprofibrate, like other peroxisome proliferators Â«o no o M l (27, 39), does not substantially elevate the activity of heat-stable N M L Â» 8 N M LP S mitochondrial enoyl-CoA hydratase activity. Administration of MCLATIVI OISTRHUTION OF MOTI l N (%) antioxidant ethoxyquin to rats fed ciprofibrate did not significantly Chart 1. Subcellulardistribution of the relative specificactivities of catalase(Ja, alter the ciprofibrate-inducible elevations in hepatic peroxisomal 2a. 3a, and 4a) and the peroxisomal palmitoyl-CoA oxidation system (Ib, 20, 3i>, and 4o) in the livers of control rats (la and Jo) and rats treated for 6 weeks with: enzyme activities (Table 1). ciprofibrate (0.1%) (2a and 2o); BHT (0.7%) (3a and 30): ciprofibrate (0.1%) (4a); Table 2 shows the effects of feeding BHA and BHT, 2 struc and BHT (0.7%) (4a and 4b). Homogenates were fractionated according to the turally different antioxidants, on specific activities of the peroxi method of Baudhuin e( al. (5). The ordinales represent the distribution of specific activities in subcellular fractions in normal relative to the highest activity in the somal marker enzyme catalase and the peroxisomal palmitoyl- ciprofibrate and in BHT relative to the highest activity in the ciprofibrate:BHT. The CoA oxidation system in rat liver homogenates. Although BHA abscissas indicate the relative distribution of protein content of nuclear (W),mito or BHT per se did not significantly elevate the activities of these chondrial(M). light mitochondrial (L), microsomal(P),and supernatant (S)fractions. 2 peroxisomal enzymes, they did not prevent the increases in the activities of these enzymes when administered in combina and BHT-treated rat liver, the highest activities of catalase and tion with ciprofibrate. In this study, the short-term treatment with peroxisomal palmitoyl-CoA oxidation system were found in the the antioxidants BHA and BHT at the concentrations of 0.7 and heavy and light mitochondrial fractions (Chart 1). As illustrated, 0.5%, respectively, did not show any signs of toxicity. the fractionation of livers of rats fed ciprofibrate (0.1%) alone Effect of Ciprofibrate and Antioxidant BHT on Subcellular (Chart 1, 2a and 2Â£>)orin combination (Chart 1, 4a and 4b), with Distribution of Catalase and Palmitoyl-CoA Oxidation System. BHT (0.7%) gave results similar to those obtained with control The results of cell fractionation are shown in Chart 1. In normal rats or rats treated only with BHT. The catalase and peroxisomal

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(3-oxidation system were markedly elevated by administration of 1234 5 6 7 8 9 10 11 12 13 14 ciprofibrate; this increase was not inhibited by concurrent BHT feeding. The catalase activity of supernatant in the livers of ciprofibrate- and ciprofibrate:BHT-fed rats was considerably higher than that in control or BHT-fed rats. The large particle fractions prepared from control, BHT-, cip rofibrate-, and ciprofibrate:BHT-treated rat livers were fraction ated by sucrose density gradient centrifugation to determine whether this antioxidant alters the density pattern of peroxisomes proliferated under the influence of ciprofibrate. As illus trated in Chart 2, BHT administration caused a subtle change in the density distribution of organelles containing catalase and peroxisomal /3-oxidation system in rats fed ciprofibrate. This resulted in a slight preponderance of peroxisomes in the heavy mitochondrial fraction (Chart 2). ...in: Â¡n SDS:Polyacrylamide Gel Electrophoretic Analysis of Large â€¢ttstselii Particle Fractions. From previous studies (27, 43), it is clear that proliferation of peroxisomes in liver cells and kidney cortical epithelium induced by peroxisome proliferators, such as ciprofi brate, results in a marked increase in the quantity of a M, 80,000 protein. In this study, we analyzed the large particle fractions of liver by SDS-polyacrylamide gel electrophoresis to determine whether antioxidants cause any alteration in the inducibility of this protein by ciprofibrate (Fig. 1). Administration of antioxidants ethoxyquin (Fig. 1, Slots 11 and 12), BHA (Fig. 1, Slots 5 to 7), Fig. 1. SDS:polyacrylamide slab gel electrophoretlc profiles of proteins in large- particle fractions obtained from the livers of normal rats and rats treated with or BHT (not illustrated) did not increase the concentration of this ciprofibrate and/or antioxidants. Large-particle fractions were prepared from the Mr 80,000 protein. As expected, ciprofibrate caused a marked livers of rats and electrophoresed as described in the text. Slots 1 and 2, normal increase in the content of this protein (Fig. 1, Slots 3 and 4), and rats; Slots 3 and 4, rats fed ciprofibrate (0.1%) in the diet for 6 weeks; Slots 5 to 7. rats fed BHA (0.5%); Slots 8 to 70, rats fed ciprofibrate (0.1 %):BHA (0.5%); Slots this increase was not prevented by concurrent administration of n and 72, rats fed ethoxyquin (0.5%); and Stofs 73 and 74, rats fed ciprofibrate antioxidants BHA (Fig. 1, Slots 8 to 70), ethoxyquin (Fig. 1, Slots (0.1%):ethoxyquin (0.5%). The protein concentration was 20 fig/slot. Arrow, posi 13 and 14), or BHT (not illustrated). tion of the M, 80,000 polypeptide. There is an increase in the amount of this peroxisome proliferation-associated protein in the livers of rats fed ciprofibrate Morphological Changes. Dietary administration of antioxi either alone or in combination with an antioxidant. dants alone caused mild to moderate increases in the amount of smooth endoplasmic reticulum in liver parenchymal cells (Fig. 3). These antioxidants exerted no perceptible alterations in peroxi somes when compared to normal rats. The liver cells of rats fed i.oo ciprofibrate (0.1%) contained numerous peroxisomes (Fig. 2). > Similar increases in peroxisome population were also encoun P tered in liver parenchymal cells of rats fed ciprofibrate in combi < 0.50 nation with an antioxidant (Fig. 4). Morphometric analysis (Table 3) demonstrated that ciprofibrate caused an 8-fold increase in

Table 3 Morphometric analysis of effects of antioxidants on ciprofibrate-inducible peroxisome proliferation in male rat liver I.OOr Male F344 rats were fed ciprofibrate (0.1%) alone or in combination with one of the antioxidants for 6 weeks. The dietary concentrations of antioxidants used in these studies were as follows: ethoxyquin (0.5%); BHA (0.5%); and BHT (0.7%). Thirty electron micrographs of randomly selected areas of liver cell cytoplasm from each group (3 animals/group, 10 micrographs/animal) were subjected to morpho- OSO metric measurement as described by Weibel (62). Points overlying cytoplasm, mitochondria, and peroxisomes were determined to obtain the volume density of mitochondria and peroxisomes. densityGroupControl Volume

IO 20 30 Â±0.85a FRACTION VOLUME (ml) Â±0.55 Ethoxyquin 18.73Â± 1.52 2.1 7 Â±0.28 Chart 2. Fractionation of livers of normal (â€¢),ciprofibrate (0.1 %) (D), BHT (0.7%) 2.60 + 0.10 P), and ciprofibrate (0.1%):BHT (0.7%) (S) -treated male rats. The large particle BHA 18.06 Â±2.65 BHT 16.82Â± 1.20 2.57 Â±0.25 fractions were sedimented into linear sucrose density gradients The distribution of Ciprofibrate 22.95 Â±1.57 18.78 Â±2.8" the activities of catalase (A) and the peroxisomal pi-oxidation system (B) shows a 21. 09 Â±3.46" Ciprofibrate + ethoxyquin 19.70 + 2.79 remarkable increase in the concentration of organelles containing these enzymes 22.92 Â±3.6" Ciprofibrate + BHA 20.72 Â±2.50 in rats treated with ciprofibrate alone or in combination with BHT relative to controls 24.5 Â±2.69" Ciprofibrate + BHTMitochondria20.50 19.5 Â±1.00Peroxisomes2.40 receiving chow or BHT. The abscissas indicate the fraction volume from the top of the gradient; the ordinales represent the relative distribution of enzyme activities â€¢MeanÂ±S.D. 0 Significantly different from controls and from animals treated only with an in fractions in normal relative to the highest activity in the ciprofibrate and in BHT relative to the highest activity in the ciprofibrate:BHT. antioxidant (p< 0.001).

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1983 American Association for Cancer Research. N. D. Lalwani et al. Â¿O fold increase in the rate of palmitoyl-CoA-dependent H202 gen 30 eration has been noted in the liver of rats fed nafenopin (22). Increased steady-state levels of H202 production were also r= 0.267 f =CU07 20 detected in the livers of rats fed Wy-14,643 (26) and bezafibrate 10 (13). There is considerable evidence which indicates that H202 causes base destruction, strand breakage, and cross-linking in isolated DMA (35), as well as induces DMA scissions in intact prokaryotic and eukaryotic cells (40). Chromatid breaks, chro- 40 matid exchanges, and unscheduled DNA synthesis have been 30 r =0.230 r =0.369 observed in mammalian cells when cultured in medium containing (SI H2O2 (58). Furthermore, a recent report by Ito ef al. (23) dem Iti onstrates the ability of p.o.-administered H2O2 to induce duo O 10 â€” denal hyperplasia and duodenal carcinoma in mice. Therefore, it oX O is conceivable that the increased generation of H2O2by prolifer oc ated peroxisomes in the liver of animals treated with peroxisome 40 proliferators causes DNA injury similar to that occurring in tissues LL o 30 r =0.265 r = exposed to ionizing radiation (37). While it is known that H2O2is formed in excess in the liver as a consequence of continued o 20 peroxisome proliferation (13, 22, 26, 29), it is not apparent at 10 present that H2O2and other free radicals are directly involved in hepatocarcinogenesis by these peroxisome proliferators. tu O er To test the hypothesis that the carcinogenicity of peroxisome proliferators is due to lipid peroxidation produced as a result of 403020toâ€¢9,|jrujir=0.2l8I the ability of these compounds to induce H2O2-generating per r =0.317 oxisome oxidases in liver (41, 44), it appears necessary to systematically examine the effects of antioxidants on hepatic effects of carcinogenic peroxisome proliferators. The antioxi nr_jj"L dants are widely used as food preservatives in order to retard the autoxidation of fat by means of their radical scavenger 0 0.2 0.4 0.6 08 1.0 0 0.2 0.4 0.6 properties (8). Ethoxyquin, BHA, and other antioxidants have RADIUS (/um) been found to inhibit the carcinogenic effects of several genotoxic chemical carcinogens that require metabolic activation (34, 60, Charts. Histogram presenting hepatic peroxisome profile radii of control (a), ciprofibrate (0.1%) in diet (o), BHT (0.7%) in diet (c), BHT (0.7%):ciprofibrate (0.1 %) 61 ). This inhibition is attributed to a decrease in the conversion (d), BHA (0.5%) (e), BHA (0.5%):ciprofibrate (0.1%) (f), ethoxyquin (0.5%) (g), and of a proximate carcinogen to the ultimate electrophilic form(s) ethoxyquin (0.5%):ciprofibrate (0.1 %) (ft) treated rats. Radii were estimated directly and, in part, to the enhancement of intracellular levels of gluta- from photographs and taken as the geometric mean of the longest and shortest thione S-transferase, an enzyme that catalyzes the binding of axis of transection. Approximately 200 transections of peroxisomes were measured for each group. various electrophiles to noninformational molecules in the cell such as glutathione (6, 16, 24, 28, 49, 50). There is virtually no information as to whether antioxidants similarly inhibit carcino the volume density of peroxisomes but no significant change in genesis by compounds such as 2,3,7,8-tetrachlorodibenzo-p- the volume density of mitochondria. The magnitude of increase dioxin, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane, and perox in peroxisome volume density was not prevented by concurrent administration of the antioxidant ethoxyquin, BHA or BHT. Die isome proliferators that do not appear to yield electrophilic or tary administration of antioxidants alone caused no significant mutagenic metabolites (3). The suggestion that the carcinoge quantitative alterations in the mitochondria and peroxisomes of nicity of these compounds is due to their ability to initiate lipid liver cells. The size distribution of peroxisomes in the liver cells peroxidation chain reactions either directly or indirectly (2, 41) implies that antioxidants might exert a protective effect. of rats in various experimental groups is illustrated in Chart 3. The studies described in this paper are part of a continuing effort to elucidate the mechanism(s) by which several structurally diverse chemicals with peroxisome-proliferative property induce DISCUSSION liver tumors. The peroxisome proliferator carcinogenesis model The mechanism(s) by which peroxisome proliferators, a group system is also best suited to examine the protective role, if any, of structurally diverse chemicals, initiate carcinogenesis is not of antioxidants against chemical carcinogens that are presum evident, but because of their ability to induce hepatic peroxisome ably non-DNA damaging. The results of the present experiments proliferation, we postulated that the liver carcinogenesis is linked demonstrate that the antioxidants (ethoxyquin, BHA, or BHT) to the endogenous metabolic disturbance(s) emanating from exert no inhibitory effect on the hepatic peroxisome proliferation sustained increase in the hepatic peroxisome population (41,44, inducible in rats by the hypolipidemic chemical ciprofibrate. Cip 59). On this premise, it was suggested that the first dehydrogen- rofibrate, like other potent peroxisome proliferators (41), induces ation step of the peroxisomal fatty acid ,8-oxidation system, hepatocellular carcinomas in rats and mice.6 Ciprofibrate pro which involves the reduction of O2 to H2O2(9), leads to an excess duced a remarkable increase in the numerical and volume density of endogenous H202 production in the livers of rats fed peroxi of hepatic peroxisomes, but it had no effect on the mitochondrial some proliferators, thereby initiating carcinogenesis. A several- relative volume. The average radius of liver peroxisomes in

1684 CANCER RESEARCH VOL. 43

ciprofibrate-treated animals was greater than that of peroxi- 1966. somes in the liver cells of control rats and rats fed only antioxi- 9. Cooper, T. G., and Beevers, H. rf-oxidation in glyoxysomes from castor bean endosperm. J. Biol. Chem., 244: 3514-3520,1969. dants in the diet. The antioxidants failed to modify these mor 10. Douglas, J. F., and Hartwell, W. V. Carcinogen bioassay of di-(2-ethyl- phological alterations induced by ciprofibrate. The results of hexyl)phthalate (abstract). Toxicologist, 1: 129, 1981. these short-term experiments also demonstrate that the antiox- 11. Eldred, G. E., Miller, G. V., Stark, W. S., and Feeney-Burns, L. Lipofuscin: resolution of discrepant fluorescence data. Science (Wash. D. C.), 276: 757- idant ethoxyquin, when fed simultaneously with ciprofibrate, 759, 1982. does not alter the inducibility of M, 80,000 polypeptide, catalase, 12. Fitzgerald, J. E., Sanyer, J. L., Schardein, J. L., Lake, R. S., McGuire, E. J., and de la Iglesia, E. A. Carcinogen bioassay and mutagenicity studies with the carnitine acetyltransferase, enoyl-CoA hydratase, and the per- hypolipidemic agent gemfibrozil. J. Nati. Cancer Inst., 67:1105-1116,1981. oxisomal palmitoyl-CoA oxidation system. The other 2 antioxi 13. Foerster, E-C., FÃ¤hrenkemper, T., Rabe, U., Graf, P., and Sies, H. Peroxisomal fatty acid oxidation as detected by H202 production in intact perfused rat liver. dants (BHA and BHT) also had no effect on the extent of Biochem. J., 796: 705-712, 1981. induction by ciprofibrate of M, 80,000 polypeptide catalase and 14. Fritz, I. B., Schultz, S. K., and Spere, P. A. Properties of partially purified the palmitoyl-CoA oxidation system. The inability of these an carnitine acetyltransferase. J. Biol. Cfiem., 238: 2509-2517, 1963. 15. Goldfischer, S., Villaverde, H., and Forschirm, R. The demonstration of acid tioxidants to inhibit peroxisome proliferation and peroxisomal hydrolase, thermostable reduced diphosphopyridine nucleotide tetrazolium enzyme activities induced by ciprofibrate suggests that they do reducÃase,and peroxidase activities in human lipofuscin pigment granules. J. not interfere with whatever mechanism(s) by which ciprofibrate Histochem. Cytochem., 14: 641-652, 1966. 16. Grantham, P. H., Weisburger, J. H., and Weisburger, E. K. Effect of antioxidant and possibly other peroxisome proliferators induce hepatic per butylated hydroxytoluene (BHT) on the metabolism of the carcinogens W-2- oxisome proliferation. Implicit in this suggestion is that antioxi fluorenylacetamide and W-hydroxy-N-2-fluorenylacetamide. Food Cosmet. dants may not alter the metabolism of peroxisome proliferators. Toxicol., 77:209-217, 1973. 17. Halliwell, B. Biochemical mechanisms accounting for the toxic action of oxygen Because peroxisome proliferation is not inhibited by antioxi on living organisms: the key role of Superoxide dismutase. Cell Biol. Int. Rep., dants when fed with ciprofibrate, it is reasonable to assume that 2: 113-129, 1978. 18. Harman, D. The aging process. Proc. Nati. Acad. Sei. U. S. A., 78:7124-7128, excess levels of intracellular H2O2 are generated as a result of 1981. increased activity of the peroxisomal fatty acid /3-oxidation sys 19. Hartig, F., Stegmeier, K., and Hebold, G. Study of liver enzymes: peroxisome tem. How effectively the H2O2and possibly other oxygen radicals proliferation and tumor rates in rats at the end of carcinogenicity studies with bezafibrate and clofibrate. Ann. N. Y. Acad. Sci., 386: 464-467, 1982. so generated are scavenged by the concurrently administered 20. Hayashi, H., HiÃ±o,S., Yamasaki, F., Watanabe, T., and Suga, T. Induction of antioxidant remains to be determined (1, 31). Long-term studies peroxisomal enzymes in rat liver by the hypolipidemic agent LK-903. Biochem. are required to determine the effects of antioxidant feeding on Pharmacol.. 30: 1817-1822, 1981. 21. Hess, R., Staubli, W., and Riess, W. Nature of the hepatomegalic effect the lipofuscin accumulation in liver cells and on the development produced by ethyl-chloro-phenoxyisobutyrate in the rat. Nature (Lond.), 208: of liver tumors in animals fed a peroxisome proliferator. In 856-858. 1965. 22. Inestrosa, N. C., Bronfman, M., and Leighton, F. Detection of peroxisomal conclusion, these experiments provide a model system to test fatty acyl-coenzyme A oxidase activity. Biochem. J., 782: 779-788, 1979. the hypothesis that peroxisome proliferator carcinogenesis is 23. Ito, A., Watanabe, H., Naito, M., and Naito, Y. Induction of duodenal tumors mediated by H2O2 and possibly other oxygen intermediates in mice by oral administration of hydrogen peroxide. Gann, 72:174-175,1981. 24. Kahl, R. Antioxidants and carcinogen metabolism. Trends Pharmacol. Sci., 3: resulting from peroxisome proliferation. Inherent in this hypoth 72-74, 1982. esis is the assumption that antioxidants by inhibiting lipid per- 25. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head oxidation could retard or inhibit this process. of bacteriophage T4. Nature (Lond.). 227: 681 -685, 1970. 26. Lalwani, N. D., Reddy. M. K., Qureshi, S. A., and Reddy, J. K. Development of hepatocellular carcinomas and increased peroxisomal fatty acid fi-oxidation ACKNOWLEDGMENTS in rats fed (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) in the semi-purified diet. Carcinogenesis, 2: 645-650, 1981. The authors wish to thank Dr. H. P. Drobeck, Sterling-Winthrop Research 27. Lalwani, N. D., Reddy, M. K., Qureshi, S. A., Sirtori, C. R., Abiko, Y., and Institute, Rensselaer, N. Y., for helpful discussions and for the generous supply of Reddy, J. K. 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Fig. 2. Male rat fed ciprofibrate (0.1% in diet), a hypolipidemic peroxisome proliferator, for 6 weeks. There is a striking increase in the number of peroxisomes (p) in liver cell cytoplasm, x 14,000.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1983 American Association for Cancer Research. Fig. 3. Male rat fed BHT (0.7% in diet) for 6 weeks. An electron micrograph of liver parenchymal cell reveals focal areas of proliferation of smooth endoplasma reticulum (ser). Peroxisome (p) complement is unchanged, x 13,750.

Fig. 4. Male rat fed ciprofibrate (0.1%) and BHT (0.7%) concomitantly in the diet for 6 weeks. The feeding of the antioxidant BHT (and also ethoxyquin and BHA) did not alter the ciprofibrate-induced hepatic peroxisome proliferation as evidenced by the presence of numerous peroxisomes (p) in this electron micrograph of a liver cell, x 11,250. 1687 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1983 American Association for Cancer Research. Noninhibitory Effect of Antioxidants Ethoxyquin, 2(3)-Tert -butyl-4-hydroxyanisole and 3,5-Di-tert-butyl-4-hydroxytoluene on Hepatic Peroxisome Proliferation and Peroxisomal Fatty Acid β-Oxidation Induced by a Hypolipidemic Agent in Rats

Narendra D. Lalwani, M. Kumudavalli Reddy, Saeed A. Qureshi, et al.

Cancer Res 1983;43:1680-1687.

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