peroxisome proliferation and lipid peroxidation in rat liver1 · ' this investigation was...

[CANCER RESEARCH 46, 1324-1330. March 1986]

Peroxisome Proliferation and Lipid Peroxidation in Rat Liver1

Sudhir K. Goel, Narendra D. Lalwani, and Janardan K. Reddy2

Department ol Pathology, Northwestern University Medical School, Chicago, Illinois 60671

ABSTRACT

Male F344 rats were fed a diet containing the peroxisomeproliferators 2-[4-(2,2-dichlorocyclopropyl)phenoxy]-2-methyl-propionic acid [ciprofibrate (0.025%)] or [4-chloro-6-(2,3-xyli-dino)-2-pyrimidinylthio]acetic acid [Wy-14643 (0.1%)] for up to

14 months to determine whether hepatic peroxisome proliferation caused by these agents results in the induction of membranelipid peroxidation in the liver. Peroxidative damage of membranelipids from whole liver, postnuclear, heavy-particle, microsomal,

and nuclear membranes was evaluated by determining the extentof formation of conjugated dienes (ultraviolet absorption, 233nm). Increased generation of diene conjugates was noted inwhole-liver, postnuclear, and heavy-particle membrane lipids of

rats fed peroxisome proliferators for 6 months or longer whencompared to controls. An additional, more intense absorptionprofile in the ultraviolet absorption range of ~276 nm was noted

in the membrane lipids derived from whole liver, postnuclear,and heavy particle pellets, but not in the nuclear and microsomalmembrane lipids of livers with peroxisome proliferation. Althoughthe exact chemical nature of this A276nmpeak is not clear, it isattributed to the formation of ketone dienes and/or conjugatedtrienes. The excess lipid peroxidation correlates with the previous observation of accumulation of abundant quantities oflipofuscin in hepatocytes of rats chronically exposed to peroxisome proliferators. The generation of conjugated dienes andketone dienes and/or trienes together with increased levels ofH2O2generation by peroxisomal enzymes, and decreased levelsof hepatic glutathione peroxidase, glutathione reducÃase, andglutathione-S-transferases, enzymes responsible for the defense

against H2O2damage, suggest the occurrence of membrane lipidperoxidation and oxidative stress in livers of rats treated withcarcinogenic peroxisome proliferators.

INTRODUCTION

Currently, several structurally dissimilar hypolipidemic drugsand certain phthalate ester plasticizers constitute two majorcategories of carcinogenic peroxisome proliferators (1-6). These

two classes of chemicals play an important role in our societytoday, the hypolipidemic agents in the control of hyperlipidemia,a major risk factor for coronary heart disease, and the phthalateester plasticizers in the formulation and manufacture of highlyversatile poly vinyl plastics. It is also expected that new classesof peroxisome proliferators will most likely be identified becauseof the current emphasis on the development of nonmutagenicchemicals for use as pharmacological, industrial, herbicidal, and

Received 8/13/85, revised 11/6/85; accepted 11/7/85.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This investigation was supported by NIH Grants CA 32504 and GM 23750.2To whom requests for reprints should be addressed, at the Department of

Pathology. Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611.

pesticidal agents, since such nonmutagenic agents are nowscreened for peroxisome proliferative activity. In view of thewidespread use of peroxisome proliferators, it is imperative tounderstand their effects on biological systems and delineate theunderlying mechanisms of toxicity and carcinogenicity. It is becoming increasingly evident that peroxisome proliferators inducea very high incidence of hepatocellular carcinomas in rats andmice (4, 7-9) although they are neither mutagenic in Ames

assays (10, 11) nor DMA damaging (12, 13). This led to thehypothesis that their carcinogenicity is related to biologicallyactive products of the proliferated peroxisomes rather than to adirect chemical effect on cellular macromolecules (4, 8). Therefore, the proposed relationship between persistent proliferationof peroxisomes and hepatocarcinogenicity implies that peroxisome proliferator induced increase in the synthesis of H2O2generating peroxisomal ÃŸoxidation enzyme system can causeprofound intrahepatic oxidative stress leading to endogenousinitiation of neoplastic change in liver (8, 14). This mechanism isconsistent with the current recognition that cellular prooxidantstates can modulate gene expression, differentiation, aging, andneoplastic transformation (15,16).

In previous studies we have shown accumulation of abundantquantities of autofluorescent lipofuscin in liver during hepatocar-cinogenesis by peroxisome proliferators (14, 17). Lipofuscin iswidely accepted as the end product of a free radical-inducedoxidative polymerization reaction involving proteins and lipids(18,19). Therefore, it appeared necessary to investigate whetherxenobiotic-induced peroxisome proliferation leads to oxidationof membrane fatty acids, initiating lipid peroxidation. In this reportwe present evidence of increased lipid peroxidation in rat liverswith peroxisome proliferation induced by ciprofibrate,3 a peroxi

some proliferator (20). The data on hepatic H2O2generation andon the levels of enzymes which play a role in the regulation ofdefenses against oxidative stress are also included.

MATERIALS AND METHODS

Chemicals. The hypolipidemic peroxisome proliferators used in thisstudy were generous gifts from the following sources: ciprofibrate (purity,>99.99%) from Sterling Winthrop Research Institute, Rensselaer, NY;and Wy-14643 (purity, >99.9%) from Wyeth Laboratories, Inc., Radnor,PA. NAD+, coenzyme A, and palmitoyl-CoA were obtained from SigmaChemical Co., St. Louis, MO. [1-14C]Palmitolyl-CoA (specific activity, 59

mCi/mmol) was obtained from Radiochemical Center Corp., Amersham,Arlington Heights, IL. All other chemicals were obtained from sourceslisted elsewhere (21, 22).

Animals and Treatment. Male F-344 rats weighing 100-150 g were

obtained from Charles River Breeding Laboratories, Wilmington, MA.They were housed in individual cages on a 12-h light, 12-h dark photo-

period in the containment facility of the center for Experimental AnimalCare of the Northwestern University Medical School, and had free access

3The abbreviations used are: ciprofibrate, 2-[4-(2,2-dichlorocyclopropyl)-phenoxy)-2-methylpropionic acid; Wy-14643, [4-chloro-6-(2,3-xylidino)-2-pyrim-idinylthio]acetic acid; GSH, glutathione; GST, glutathione-S-transferase; GSH-P,glutathione peroxidase; GSH-R, glutathione reductase.

CANCER RESEARCH VOL. 46 MARCH 1986

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PEROXISOME PROLIFERATION AND LIPID PEROXIDATION

to water and food. Peroxisome proliferators were administered in powdered rat chow at concentrations indicated. The diets were mixedthoroughly in a mechanical mixer, once weekly, and stored in a refrigerator. The daily consumption of diet by each rat was estimated at -10-

14 g. Control rats were fed rat chow without the drug. At the end of thetreatment period the animals were killed under light ether anesthesia andthe livers were removed and processed as described below.

Subcellular Fractionation of Liver. The livers were homogenized(10% homogenate, w/v) in ice-cold 0.25 M sucrose containing 0.003 MEDTA (pH 7.4) using a Potter-Elvehjem homogenizer. The homogenates

were fractionated into heavy particle (mitochondria, peroxisomes, andlysosomes), postnuclear (mitochondria, peroxisomes, lysosomes, andmicrosomes), and microsomal pellets according to the method outlinedby Kurup ef al. (23) as described before (24). Nuclei were obtained bythe method of Bresnick ef al. (25) as modified by Patton et al. (26). Thesubcellular fractions so obtained were suspended in 0.25 M sucrosecontaining 0.003 M EDTA and were used for lipid extraction eitherimmediately or after storage at -70Â°C.

Extraction of Lipids and Detection of Conjugated Dienes. Lipidsfrom various subcellular fractions were extracted in 20 ml of chloro-

fornrmethanol (2:1) according to the method of Bligh and Dyer (27).Lipids in the chloroform phase were recovered by evaporating thechloroform under oxygen-free N2 (ultrapure carrier, Grade 99.99% N2).

The final total lipid extract was dissolved in cyclohexane (1 mg/ml) andthen scanned from 300 to 220 nm against a cyclohexane blank in aBeckman Model 25 spectrophotometer. Total lipid content was measured by the method of Chiang ef al. (28). Lipids extracted from liverpostnuclear membranes of rats treated with carbon tetrachloride servedas a positive control for detection of diene conjugates (29).

Measurement of H2O2.The basal levels of H2O2in liver homogenateswere measured by the ferrithiocyanate method as described by Thurmanef al. (30). Liver homogenates (5%, w/v) were prepared in 0.25 M sucrosesolution containing 1 mivi sodium azide. Proteins were precipitated bycold 50% trichloroacetic acid. To 1 ml of supernatant, 0.2 ml of 10 mwferrous ammonium sulfate and 0.1 ml of 2.5 M potassium thiocyanatewere added, and the absorption of red ferrithiocyanate complex formedin the presence of H2O2 was measured at 480 nm against appropriateblanks, using Beckman Model 25 spectrophotometer. The values areexpressed as nmo\ H2O2 present per g liver. The substrate-mediated

generation of H2O2 by peroxisomal /i oxidation system was measuredby incubating aliquots of liver homogenates in 50 mw Tris HCI, pH 8.3,containing NAD* (0.2 HIM); CoA (0.1 HIM); flavin adenine dinucleotide

(0.01 HIM); dithiothreital (0.01 HIM); Triton X-100 (0.1%); bovine serum

albumin (0.0075%); MgCI2 (5 ITIM);ATP (2.5 rriM); sodium azide (1 HIM),and substrate palmitate (15 ^M) in a total reaction mixture of 1.0 ml. Thereaction mixture was incubated at 37Â°C for 40 min. The reaction was

terminated by adding 50% cold trichloroacetic acid to get a final concentration of 5%. The hydrogen peroxide was measured by the ferrithiocyanate method as described above.

Enzyme Activities. Enzymatic activities in liver homogenates weremeasured as follows: peroxisomal palmitoyl-CoA oxidizing activity by the

method described by Neat and Osmundsen (31) using antimycin A (10/jg/ml) to block ÃŸoxidation due to mitochondria; catalase was measuredby the method of Luck (32) as described elsewhere (33). GSH contentwas determined by the method of Cohn and Lyle (34) as modified byHissin and Hilt (35); GST was assayed according to the procedure ofHabig ef al. (36) with 1,2-dichloro-4-nitrobenzene and 1-chloro-2,4-dini-trobenzene as substrates; GSH-P was measured using GSH and H2O2

as substrates according to the procedure described by Hafeman ef al.(37), and GSH-R was assayed according to the method outlined by

Racker (38). Protein concentration was determined by the method ofLowry ef al. (39), using bovine serum albumin as a standard.

Electron Microscopy. Postnuclear and heavy particle pellets obtainedfrom livers of rats exposed to ciprofibrate for 6 months or longer werefixed in 2% OsO4, dehydrated, and embedded in Epon. Semithin sectionswere examined by a light microscope. Ultrathin sections cut from selected

regions showing a uniform layer of particles were stained with lead citrateand examined in an electron microscope.

RESULTS

Lipid Peroxidation. Lipid peroxidation in membranes obtainedfrom whole-liver homogenates and in membranes of subcellular

fractions was compared in rats fed control and peroxisomeproliferator-containing diets. Since the major sites of lipid per

oxidation damage within the cell are in the biomembranes ofsubcellular organelles, it appeared necessary to compare theextent of lipid peroxidation damage in microsomal, nuclear, large-particle, and postnuclear membranes. Lipid peroxidation-induceddouble-bond rearrangements in the hydrocarbon chain of poly-

unsaturated fatty acids result in the appearance of conjugateddienes which exhibit characteristic UV absorption pattern at 233nm (40). Lipids extracted from isolated membranes of whole liverhomogenates of rats fed ciprofibrate (0.025%, w/w) and Wy-14643 (0.1%, w/w) for 6 months yielded a clear peak of conjugated dienes at 233 nm, when compared to rats fed a regulardiet (Fig. 1). In addition, in the membrane lipids from ciprofibrate-and Wy-16463-treated rats, a second peak was noted in the 276

nm range (Fig. 1). Analysis of UV spectra of extracted lipids frompostnuclear pellets (Fig. 2) and heavy-particle pellets (Fig. 3) of

rats fed ciprofibrate for 6 months also revealed two distinctpeaks, one at 233 and the other at 276 nm. Such increases werenot evident in lipids derived from microsomal pellets. As can beseen from Fig. 2, the 276 nm peak was not prominent either incontrol or in carbon tetrachloride-treated rat liver postnuclear

pellets. The 233 and 276 nm peaks were only slightly prominent

220 240 260 28O

WAVELENGTH(nm)

Fig. 1. UV absorption patterns of lipidsextracted from membranesderived fromwhole liver homogenates of rats fed (a) control diet; (b) Wy-14643 (0.1%, w/w);and (c) ciprofibrate (0.025%, w/w) in chow for 6 months. Each trace representsthe meanabsorption pattern of lipidsamplesobtained from 3-5 rats. The absorptionat -233 nm indicates the presence of conjugated dienes, and the differencespectrum (Â±ABS233)is obtained by subtracting the mean values of control spectrafrom the mean values of the spectra of peroxisome proliferator-treated rats. Anadditional peak at -276 nm, which is more intense in peroxisome proliferator-treated animals, is suspected to indicate the presence of conjugated trienes and/or ketone dienes, although proteolipids rich in tyrosine and tryptophan may alsogive absorbance in the region of -280 nm (41, 42).


1325




&ABStn*OJU(M; OIICIAABS276 =0 Ofllb), O 45ICI

220 260 300

WAVELENGTH (nm)

Fig.2. UV absorption patterns of lipids extracted from membranesfrom post-nuclear pellets, which contain all membranes except nuclear membranes of ratsfed (a)control diet; and (c) ciprofibrate (0.025%, w/w) in chow for 6 months. Thespectrum (6) represents the profile of lipids derived from the liver of a rat treatedwith CCU (0.25 ml/100 g body wt) by gavage and killed 16 h later. Note theincreasedabsorption of -233 nm in ciprofibrate- and CCU-treatedrats (AASS233),indicating increased lipid peroxidation. The increase in absorption at ~276 nm isvery striking in ciprofibrate-treated animals, when compared to control and CCU-treated rats.

"220 240 260 280

WAVELENGTH (nm)

300

Fig.3. UV absorption patterns of lipids derived from membranes of heavyparticle pellets (composed predominantlyof mitochondria, peroxisomes, and lyso-somes)obtained from rats fed (a)control chow, and (o) ciprofibrate (0.025%, w/w)in rat chow for 6 months. For other details about the difference spectrum ofabsorption at 233 and 276 nm, see the legend for Fig. 1.

in postnuclear-pellet lipids of rats fed ciprofibrate for a short

duration of 4 weeks (Fig. 4). In comparison, the 276 nm peakwas very large in the lipids extracted from postnuclear pellets(Fig. 5) prepared from nontumorous portions of liver from ratsbearing hepatocellular carcinomas induced by chronic administration of ciprofibrate or Wy-14643 (Fig. 6). This 276 nm peak,

however, was only slightly intense in the lipids of highly purified

nuclear pellets (Fig. 7) and microsomes (not illustrated) of per-oxisome proliferator-treated rats. As illustrated in Fig. 5, the

postnuclear pellet contains numerous lipofuscin pigment profiles,abundant peroxisomes, and several mitochondria. The highlyprominent 276 nm peak in the livers of rats chronically treatedwith peroxisome proliferators most likely reflects the formationof conjugated trienes and ketone dienes which have an absor-bance in the range of 275-283 nm (40-42). It is also pertinent

to point out that proteolipids, rich in tryptophan and tyrosine,can also give intense absorbance at -280 nm (41, 42).

H2O2Generation. The basal and substrate (palmitate)-gener-ated H2O2levels were measured in the liver homogenates of ratstreated with ciprofibrate (Table 1). Basal H202 levels were higherin peroxisome proliferator-treated rat livers when compared tountreated controls. Addition of palmitate, a substrate for perox-isomal fatty acid ÃŸoxidation system, resulted in >6-fold increase

in the H2O2level (Table 1).GSH, GST, and Related Enzyme Activities. Administration

of the peroxisome proliferator, ciprofibrate, for 5 weeks resultedin a marked decrease in the activity of hepatic GST (Table 2). Incontrast, the GSH levels in the livers of ciprofibrate-treated rats

were slightly elevated (Table 2). The time course of changes inhepatic catalase, ÃŸoxidation enzyme system, and GSH and GSTlevels in rats fed ciprofibrate for up to 22 days are shown in Fig.8. Dose-response studies indicated that ciprofibrate adminis

tered at 0.001 % dietary level for 4 weeks reduced hepatic GSTlevel by more than 50%. At 0.01 and 0.02% dietary levels,ciprofibrate caused ~90% reduction in GST activity (Fig. 9). Theactivity of selenium dependent GSH-P, which utilizes H202 as

well as hydroperoxide as substrates (43, 44) was also reducedin the livers of ciprofibrate-treated rats (Table 2). Ciprofibratealso inhibited the activity of hepatic GSH-R (Table 2).

DISCUSSION

Several studies have now established that the currently identified carcinogenic peroxisome proliferators are not mutagenicand that they do not covalently interact with or damage cellular

220 260WAVELENGTH (nm)

300

Fig.4. UV absorption patterns of lipids extracted from membranes of postnuclear pellets of liver of rats fed (a)control, and (b) ciprofibrate (0.025%, w/w) for 4weeks. Note that 4-week treatment of ciprofibrate caused only a subtle increase

nmand


1326




w^wmmA v.*Â»Â»*.â€¢̂H k ~^L --^^fc f^Hv. ^ 'â€¢'/*. A T*

Â¿A'fc'ô^.. BL_ v.*S*E AÂ»,,

MO'( !*fîtÂ«

â€¢â€¢i? . ' - -^' â€¢*. yÂ»

^T^r ^/-^â€¢' â€¢->A.*â€¢**,* . i

The uninvolved liver

Â«Â«Â¿î. Â«xWÂ»Â»*rEi:Ra3Â»_^sKS*!i(?<r&,-v,Â»Ã¯;.-â€”.Â«Â¿^rÂ¿;â€¢^Â»w-..c.-.jaÂ»u-Tr -^ .ii*-

Fig. 5. Low-power survey electron micrograph of the postnuclear pellet obtained from the nontumorous portions of liver of a rat fed ciprofibrate. The uninvolved liverwas separated from the liver tumors, homogenized, and the postnuclear pellets were obtained by differential centrifugation. Note the presence of numerous peroxisomes(P), most of which show partial loss of matrix proteins. Mitochondria (M), and several profiles of lipofuscin (LI) are also seen, x 6800.

DMA (9-13). Therefore, it appears that the mechanism of carci-

nogenesis by peroxisome proliferators may be mediated by aprocess other than the direct chemical-DNA interactions. A

strong positive correlation between hepatic peroxisome proliferation and the development of hepatocellular carcinomasprompted us to postulate that the peroxisome proliferator-in-

duced liver carcinogenesis is linked to the oxidative stress emanating from sustained increase in peroxisome population andthe attendant enhanced synthesis of H2O2generating peroxiso-mal ÃŸoxidation enzymes (4, 6, 8). Although the precise mechanisms by which intracellular oxidative stress influences initiationand promotion of carcinogenesis remains unknown, it is generallyheld that H2O2and other reactive oxygen species (OH; O2~, and

102) can cause DMA damage either directly or by initiating lipid

peroxidation (15, 21, 45, 46). Alternatively, it is conceivable thatthe oxidative stress may lead to the activation of oncogene(s) ormay give rise to resistant hepatocyte population lacking peroxisome proliferator receptors. The postulated link between peroxisome proliferation-mediated oxidative stress and carcinogen-

icity implies that tumors should develop only in organs (tissues)displaying profound peroxisome proliferation in animals exposedto peroxisome proliferators.

The results of the present study demonstrate increased lipidperoxidation in the liver of rats fed for 6 months or longer a dietcontaining ciprofibrate or Wy-14643, two potent carcinogenic

peroxisome proliferators (3, 20). In contrast, the results of thesame analyses on rats fed these agents for a relatively shortperiod of 4 weeks are somewhat equivocal in that the differencespectra of less than 0.1 A units in this semiquantitative assay ofconjugated dienes is not convincing. This suggests that sustained injury to liver cells by persistent peroxisome proliferationmay be necessary to induce peroxidative damage to membranelipids. Lipid peroxidation is known to cause double bond rearrangements in the hydrocarbon chain of fatty acids (40-42, 44-

45). This results in the appearance of conjugated dienes whichabsorb intensely at 233 nm, whereas the intact polyenoic fattyacids with unconjugated double bonds absorb strongly onlybelow about 225 nm in the UV spectrum (40). Increased levels





"220 260 300

WAVELENGTH (nm)

Fig. 6. UV absorption patterns of lipids extracted from membranes of postnu-clear pellets of rats fed (a) control diet; (b) WY-14643 (0.1%, w/w); and (c)ciprofibrate (0.025%, w/w) for 14 months. The liver of ciprofibrate- and Wy-14643-treated rats contained multiple liver tumors. The postnuclear pellets were obtainedfrom portions of liver devoid of grossly visible tumors. Note the dramatic increasein the A/*SS276nm in the lipids derived from rats chronically treated with theperoxisome proliferators. This coincides with excessive accumulation of lipofuscinin these livers (14).

Om

220 260WAVELENGTH (nm)

300

Fig. 7. UV absorption patterns of lipids extracted from highly purified nuclearmembranes of rats fed (a) control diet; (b) Wy-14643 (0.1%, w/w); and (c) ciprofibrate (0.025%, w/w) for 6 months. Increases in absorbance at 233 and 276 nmare not as marked as those seen in the lipids of heavy particle and postnuclearmembranes illustrated in Figs. 2, 3, and 6.

of conjugated dienes considered as prima facie evidence of lipidperoxidation have been seen in the lipids of postnuclear andheavy-particle membrane pellets of liver of rats treated withciprofibrate or Wy-14643 for 6 months or longer. Absence of

increase in the absorbance at 233 nm in microsomal membrane

lipids observed in the present study suggests that peroxisomeproliferator-induced lipid peroxidation occurs predominantly in

the membranes of large subcellular organelles (peroxisomes,mitochondria, or lysosomes). The lipids from these membranesalso showed an additional more intense absorbance pattern at276 nm, which is generally attributed to ketone dienes and orconjugated trienes (40). Similar dramatic increase of absorbancein this region has not been reported in the literature dealing withlipid peroxidation abnormalities (40, 47, 48). The absence ofsubstantial increase in this 276 nm peak in microsomal andnuclear membrane lipids suggests that ketone diene formationmay also be restricted to the membranes of the large subcellularorganelles.

Excessive accumulation of autofluorescent lipofuscin is regularly encountered in livers of rats exposed chronically to peroxisome proliferators (14, 17). This pigment presumably reflectsmembrane damage due to biologically damaging free radicals(14, 18, 19). Peroxisomal oxidases, such as fatty acyl-CoAoxidase, urate oxidase, o-amino acid oxidase, and a-hydroxyacid oxidase reduce O2 and generate H2O2 (49, 50). The tran-scriptional activation of fatty acyl-CoA oxidase gene (51) andincreased basal and substrate-generated H2O2levels in the liversof peroxisome proliferator-treated rats are thought to be the

basis for the observed increase in lipid peroxidation in the presentstudy. Under normal conditions catalase can efficiently detoxifylow rates of H2O2 generated within the peroxisomes (52, 53).However, the high permeability of the peroxisomal membrane toH2O2(14, 52) results in the diffusion of some H2O2,even at lowrates of production. This diffusion may be accelerated in liverswith peroxisome proliferation because such peroxisomes containlow levels of catalase and increased concentration of H2O2producing ft oxidation enzymes per unit peroxisome volume (4,6). H2O2 per se or hydroxyl radicals resulting from the iron-catalyzed decomposition of H2O2 can lead to uncontrolled lipidperoxidation reaction in livers of rats with peroxisome proliferation. It is also of particular interest to note that GSH-P and GSH-

R, which are involved in H2O2 metabolism, (52) are also significantly reduced in the livers of rats treated with ciprofibrate. Thismay result in a further compromise of endogenous defenseagainst excess H2O2produced by proliferated peroxisomes.

In summary, the present study clearly demonstrates increasedlipid peroxidation in the membranes of large subcellular particlesof liver in rats chronically exposed to peroxisome proliferators.

Table 1Changes in liver weight, catalase activity, and H2O2generation in rats fed

Cipro-fibrate

Ciprofibrate was mixed in powdered rat chow (0.025%, w/w) and administeredto male F344 rats for 5 weeks. The basal and substrate-generated H2O2 levelswere determined in the whole liver homogenate. Catalase activity was measuredin the 100,000 x g supernatants of deoxycholate-treated homogenates of liver.The results are expressed as mean Â±SD of 4 to 6 rats.

HydrogenperoxideGroupControl

CiprofibrateLiver

wt(9/1 00 gbodywt)3.5

Â±0.49.3 Â±0.4aCatalase

(units/mgprotein)45

Â±394Â±9aBasal

levels(xmol/g

liver)0.522

Â±0.0180.652 Â±0.025*Generated

levels(palmitate

as substrate;(iimol/min/g

liver)0.326

+ 0.1571.922 Â±0.345a

* Significantly different from controls (P < 0.001) using Student's f test.

Significantly different from control (P < 0.01) using Student's t test.


1328




Table2Effect of ciprofibrate on glutathione and associated enzymes in rat livers

Ciprofibrate (0.025%, w/w) was mixed in powdered rat chow and fed to male F344 rats for 5 weeks. The levels of glutathione, glutathione-S-transferase, glutathioneperoxidase, and glutathione reducÃasewere determined in cytosol prepared from liver homogenates as described in "Materials and Methods." The values are mean Â±

SD of 4 to 6 rats and represent: GSH, ^g/g liver; GST, ^mol/min/mg protein using 1-chloro-2.4-dinitrobenzene (CDNB) or 1,2-dichloro-4-nitrobenzene (DCNB) assubstrates; GSH-P, ^mol NADPH oxidized using H20Z as substrate/min/mg protein; GSH-R, nmol NADPH oxidized/mm/mg protein.

GST

Group GSH CDNB DCNB GSH-P GSH-R

ControlCiprofibrate

1902 Â±1972600 Â±36"

2.06 Â±0.350.32 Â±0.06a

0.07 Â±0.0050.02 Â±0.005a

1.97 Â±0.110.716 Â±0.06a

0.52 Â±0.060.28 Â±0.02a

a Significantly different from controls (P < 0.001) using Student's f test.

1000

Fig. 8. Time course of dietary administration of ciprofibrate (0.025%, w/w inchow) on hepatic peroxisomal il oxidation enzyme system, peroxisomal catalase,GSH, and GST. As expected, ciprofibrate caused nearly a 2-fold increase in catalaseactivity and about 10-fold increase in ÃŸoxidation capacity. GSH levels were slightlyelevated, whereas the GST activity [using 1-chloro-2,4-dinitrobenzene (CDNB) and1,2-dichloro-4-nitrobenzene (DCNB) as substrates] was markedly decreased.

This observation is consistent with increased H2O2 generationand excessive accumulation of lipofuscin in hepatocytes. Increased levels of conjugated dienes (u233nm)and ketone dienes(A276nm)are indicative of lipid peroxidation damage to membranes. The intense absorbance at 276 nm, particularly in liversof rats treated for long duration may be due to the contents oflipofuscin, since such structures become abundant in the livercells of rats chronically exposed to peroxisome proliferators, andare seen in the postnuclear and heavy particle pellets used inthis study for lipid extraction (Fig. 5). The results of the presentstudy further substantiate the existence of peroxisome proliferation-associated intrahepatic oxidative stress. It is of interest to

note that increased lipid peroxidation has been observed in thelivers of rats treated with methapyrilene hydrochloride (47) andcholine-deficient diet (48), both of which induce liver tumors by

mechanisms unrelated to direct DMA damage. Additional studiesare essential to understand the interrelationships among oxidative stress, lipid peroxidation, oncogene activation, and neoplas-tic transformation in livers.

S 20I

I ,.5

1.0

0.3

abedCiprofibrate cone.

Fig. 9. Effect of various dietary levels of ciprofibrate on hepatic GST [1-chloro-2,4-dinitrobenzene (CDNB as substrate)] activity. Control diet (a) and ciprofibrate.0.001% (b). 0.01% (c), and 0.02% (d). administered for 4 weeks.

ACKNOWLEDGMENTS

We thank Dr. R. O. Recknagel for helpful sugestions. We thank MohammedUsman for excellent technical assistance and Karen McGhee for her excellentsecretarial assistance.

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1986;46:1324-1330. Cancer Res Sudhir K. Goel, Narendra D. Lalwani and Janardan K. Reddy Peroxisome Proliferation and Lipid Peroxidation in Rat Liver

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