Effects of the Hepatocarcinogen Nafenopin, a Peroxisome

[CANCER RESEARCH 45, 5011-5019, October 1985]

Effects of the Hepatocarcinogen Nafenopin, a Peroxisome Proliferator, on the Activities of Rat Liver Glutathione-requiring Enzymes and Catatase in Comparison to the Action of PhÃ©nobarbital

Kazunori Furukawa,1 Satoshi Numoto,2 Keizo Furuya,3 Noriko T. Furukawa,4 and Gary M. Williams5

Nay/or Dana Institute for Disease Prevention, American Health Foundation. Dana Road, Valhalla, New York 10595

ABSTRACT ne//a-microsome mutagenicity test (5-7), inhibition of DMA syn thesis in lymphocytes (6), and the hepatocyte primary culture- The biochemical effects in the livers of male rats of prolonged DMA repair test (8). However, it has been reported that two administration of the experimental hepatocarcinogen nafenopin, peroxisome proliferators induced unscheduled DMA synthesis in a hypolipidemic agent and peroxisome proliferator, were com hepatocytes measured by liquid scintillation counting (9). Never pared to those of another experimental liver carcinogen, phÃ©no theless, overall these agents appear not to be carcinogens of barbital, which acts as a neoplasm promoter. Feeding of nafen the DNA-reactive type but rather epigenetic agents (10), as opin, 0.03 mmol/kg basal diet for up to 24 weeks increased the proposed by Reddy and Lalwani (4). numbers of hepatic peroxisomes, increased catalase activity, The increased numbers of hepatocyte peroxisomes induced markedly decreased cytosolic glutathione transferase activities by hypolipidemic agents are accompanied by a marked increase toward two substrates, decreased cytosolic glutathione peroxi- in the peroxisomal enzymes involved in lipid metabolism (11-13). dase activities toward H2O2 and two organic peroxides, and /3-Oxidation of lipids in peroxisomes results in production of H2O2 suppressed the age-related increase in 7-glutamyl transpepti- (13) and this is probably accentuated in proliferated peroxisomes dase activity. In contrast the livers of rats fed an equimolar because the increase in the activity of the fatty acid oxidation concentration of phÃ©nobarbitaldisplayed increases in cytosolic system is disproportionally greater than that of catalase (EC glutathione transferase activities and enhancement of 7-glutamyl 1.11.1.6) activity (14), the peroxisomal scavenger enzyme that transpeptidase activity but no changes in glutathione peroxidase decomposes intraperoxisomal H2O2(15). Moreover catalase ac activities. There was also an enhancement of catalase activity tivity per unit volume of peroxisome has been shown to decrease without apparent increase in peroxisome number. Enzyme ki continuously during treatment with a hypolipidemic agent (16). netic analyses revealed that the cytosolic glutathione transferase Based on these observations the peroxisome proliferation in activities toward two halogenonitrobenzene substrates were duced by long-term administration of hypolipidemic agents has inhibited in the rats fed nafenopin and displayed elevated Kmand been suspected to result in an excessive generation of H202 in decreased Vmax.Kinetic studies of glutathione transferase activ hepatocytes (17). H2O2 can escape from peroxisomes (18) and ities in which nafenopin was mixed with normal rat liver cytosols the conversion of H202 to reactive species and the reaction of in the assay system revealed competitive type inhibition toward 1-chloro-2,4-dinitrobenzene and a noncompetitive type of inhibi the latter with DNA has been suggested to be the basis for the carcinogenicity of peroxisome proliferators (3, 4). tion toward 3,4-dichloronitrobenzene. Likewise activities of glu Outside of peroxisomes GSH6 peroxidase (EC 1.11.1.9), is a tathione peroxidases toward H202 and eumene hydroperoxide scavenger of H2O2, as well as of a variety of organic hydroper- were suppressed by in vitro addition. Thus the effects of nafen oxides (19). This enzyme therefore would play a key role in the opin and phÃ©nobarbitalon liver biochemistry were very different. decomposition of any H202 that might escape from peroxisomes The inhibition of hepatic biotransformation and scavenger sys tems by nafenopin is suggested to be relevant to its hepatocar- (18). Ciriolo et al. (20) reported that clofibrate and another cinogenicity. peroxisome proliferator, fenofibrate, lowered the activity of GSH peroxidase toward an organic hydroperoxide and reduced the activity of Superoxide dismutase (EC 1.15.1.1) suggesting an INTRODUCTION inhibiting effect of the drugs on these extraperoxisomal scaven Several hypolipidemic agents such as clofibrate and nafenopin ger enzymes which deal with oxygen radicals. Awasthi ef al. which induce peroxisome proliferation (1, 2) have caused mainly (21), however, found only minimal inhibition of GSH peroxidase liver cancer and to a lesser extent neoplasms at other sites in by ciprofibrate. This aspect requires further study because it is rodents (3, 4). Experimental carcinogens of this type have not possible that alterations of H2O2 and oxygen radical levels by displayed genotoxicity in various assays including the Salmo- hypolipidemic peroxisome proliferators could originate not only from peroxisome proliferation but also from effects on scavenger 1 Present address: Department of Pathology, Cancer Research Institute, Sap systems outside of peroxisomes. poro Medical College, Nishi-17-chome, Minami-1-jo, Chuo-ku, Sapporo 060, Japan. 2 Present address: Department of Pathology, University of Tokushima, School Another enzyme system which deals with reactive species is of Medicine, Kuramoto 3, Tokushima 770, Japan. GSH transferases (EC 2.5.1.18). GSH transferases are a group 3 Present address: Pathology Department, Ehime Prefectural Central Hospital, Kasuga-cho 3, Matsuyama, 790, Japan. of multifunctional enzymes (22) involved in detoxification of a 4 Present address: Department of Pathology, Cancer Research Institute, Sap broad spectrum of both endogenous substances and xenobiotics poro Medical College, Nishi-17-chome, Minami-1-jo, Chuo-ku, Sapporo 060, Japan. 5 Recipient of National Cancer Institute Grant CA17613-10 in partial support. To â€¢Theabbreviations used are: GSH, reduced glutathione; GGT, -y-glutamyl whom requests for reprints should be addressed. transpeptidase; ENPP, 1,2-epoxy-3-(p-nitrophenoxy)propane; GSSG, oxidized glu Received 10/26/84; revised 4/16/85; accepted 6/28/85. tathione; CDNB, 1-chloro-1,2-dinitrobenzene; DCNB, 3,4-dichloronitrobenzene.

CANCER RESEARCH VOL. 45 OCTOBER 1985 5011

including carcinogens (23-28). Liver GSH transferase activities For the measurements of catalase and GGT activities homogenates have been reported to be decreased by clofibrate (29) and in 10 vol of distilled water and of 0.2 M sodium phosphate buffer, pH 8.0, ciprofibrate (21). This effect could be of importance to the respectively, were prepared as mentioned above. All assays were carried carcinogenic properties of peroxisome proliferators because out at least in duplicate and the data were corrected for nonenzymatic GSH transferases possess selenium-independent GSH peroxi- reactions. Chemicals. GSH, ENPP, GSSG reductase (yeast, type III), eumene dase activity which reduces a variety of organic hydroperoxides hydroperoxide, tert-butyl hydroperoxide, NADPH, L-7-glutamyl-p-nitroan- (30, 31). Also GSH transferases have been postulated to inhibit ilide, and glycylglycine were purchased from Sigma Chemical Co., St. nonenzymatically the initiation of lipid peroxidation (27, 28). Louis, MO; CDNB and DCNB were from Eastman Kodak (Rochester, In a previous study we found that nafenopin inhibited the NY). activity of GGT (EC 2.3.2.2) (32), which like GSH peroxidase and Cytosolic GSH Transferase. The activities of GSH transferases were transferases is a GSH-requiring enzyme. In light of all these measured at 25Â°Cusing a Beckman Model 25 spectrophotometer with observations, we undertook the present study to determine the CDNB, DCNB, and ENPP as substrates according to the procedure of effect of nafenopin on the three GSH-requiring enzyme systems. Habig et al. (38). The reaction mixtures were identical with those reported by Habig and Jakoby (39). One unit of the enzyme activity was defined In this study we compared the effect of nafenopin on these as 1.0 nmol of GSH conjugate production/min at 25Â°C. enzymes with that of phÃ©nobarbital,which also produces rodent Cytosolic GSH Peroxidase. GSH peroxidase activities were mea liver neoplasms (33, 34) but acts as a neoplasm promoter (35, sured spectrophotometrically by a modified method of Jensen and Clau 36) in contrast to nafenopin (32, 37). We report here that feeding sen (40). The standard assay mixture (1.0 ml) consisted of 48 mw sodium of nafenopin under conditions which produce a marked hepatic phosphate buffer, pH 7.4, containing 4.8 mw EDTA-Na2, 3.75 Ã•TIMNaN3, peroxisome proliferation and elevation of catalase activity de- 5 mM GSH, 1.0 IU GSSG reductase, 0.28 mM NADPH, and 0.25 miwi creasd both cytosolic GSH peroxidase and transferase activities. H2O2(the substrate specific for selenium-dependent GSH peroxidase) or Also the age-related increase in GGT activity was inhibited. In 1.2 mM eumene hydroperoxide, or 0.07 mw tert-butyl hydroperoxide. contrast dietary phÃ©nobarbital enhanced the activities of GSH The mixture containing the cytosol fraction and the chemicals other than transferases and induced GGT activity in periportal hepatocytes, H2O2 or the hyproperoxides were equilibrated by an incubation for 10 whereas the GSH peroxidase activities were not altered. In min at room temperature. The reaction was then started with addition of one of the three peroxides. One unit of enzyme activity was defined as addition phÃ©nobarbitalelevated catalase activity without produc the amount of GSH peroxide leading to the oxidation of 1 nmol NADPH ing apparent peroxisome proliferation. The relevance of these by GSSG reductase/min at 25Â°C. observations to the liver carcinogenicity of these agents is dis Catalase. For the assay of catalase activity an aliquot of homogenate cussed. was mixed with an equal volume of 1.0% Triton X-100 and diluted with distilled water. Spectrophotometric measurement of the activity was performed by the method of Beers and Sizer (41). One unit of the enzyme MATERIALS AND METHODS activity was defined as the amount catalyzing decomposition of 1.0 /Â¿mol H2O2/min at 25Â°C. Animals and Treatments GGT. Activity in homogenates was measured according to the method Male F344 rats weighing 250-280 g were obtained from Chartes River of Novogrodsky ef al. (42). The reaction mixture was identical with that Breeding Laboratories, Kingston, NY. They were maintained in a con reported previously (43). A unit of the enzyme activity was defined as the amount catalyzing the release of 1.0 nmol p-nitroaniline/min at 25Â°C. ventional specific-pathogen-free facility fully accredited by the American Association for the Accreditation of Laboratory Animals' Care and their Protein Determination. Protein contents were measured by the Folin care conformed to the Care and Use of Laboratory Animals (NIH-78-23). method using crystalline bovine serum albumin as the standard. Enzyme Kinetics. Michaelis-Menten kinetics of liver cytosolic GSH The rats were divided into 3 groups of 10 animals each. The control group was fed NIH-07 diet (Bio-Serv, Frenchtown, NJ). The experimental transferases in nafenopin-fed and untreated rats were compared at 24 groups were fed NIH-07 diet which contained 0.093% nafenopin (kindly weeks of feeding using CDNB and DCNB as substrates. GSH concen donated by CIBA Pharmaceutical Co., Summit, NJ) or 0.07% phÃ©nobar trations in the assay systems were fixed at 1.0 and 5.0 mw for the bital (Malinckrodt Chemical Works, St. Louis, MO), which represent assays of the GSH transferase activities toward CDNB and DCNB at approximately 0.03 mmol of each compound per kg diet. Rats were 0.05 to 1.0 mM, respectively. The Km and Vâ„¢, were calculated by a computer program using least-squares estimation of a nonlinear regres given the diets and water ad libitum until the day of killing. sion curve (44). Apparent kinetics is illustrated with Lineweaver-Burk At 6 and 24 weeks, 4 and 6 rats in each group, respectively, were used for biochemical and histochemical examinations of the livers. Ani plots. mals were killed over a period of 3 h from 10:00 a.m. to 1:00 p.m. Under To examine direct actions of nafenopin on the activities of GSH anesthetization with ethyl ether animals were exsanguinated by cutting transferase and peroxidase liver cytosol fractions were obtained from 3 the abdominal inferior vena cava, after which the livers were washed free normal male F344 rats (body weight, 261 Â±18 g (SD); liver weight, 9.8 of blood by perfusion with saline through the portal vein. Â± 0.9 g). Nafenopin was added to the GSH transferase and GSH peroxidase assays as a 30% ethanol solution in the assay buffers such Biochemical Assays of the Liver Enzyme Activities that the final concentration of ethanol was 3% and the pH of the buffers was indentical to that without nafenopin. Addition of nafenopin at con Liver Preparations. Representative liver homogenates were prepared centrations higher than 3.0 mw in the assay systems led to its precipi by using portions of every lobe of each rat in each experimental group. tation. The GSH transferase activities toward CDNB and DCNB were The preparation of homogenates and cytosol fractions of liver was then measured immediately. In the case of GSH peroxidase activities the conducted at 0-4Â°C. Liver was homogenized in 10 vol of 0.25 M sucrose reaction mixtures with added nafenopin were incubated for 10 min prior containing 5 mw MgCI? and 20 rriM Tris-HCI, pH 7.6, with 10 strokes of to the addition of the peroxide substrates which started the reaction. a Potter-Elvehjem homogenizer. The homogenates were then centri- The in vitro effect of nafenopin on Michaelis-Menten kinetics of GSH fuged at 10,000 x g for 10 min followed by further centrifugation of the transferase in normal rat liver cytosol fraction was also examined. The supernatant at 105,000 x g for 60 min in a Beckman L5-75 ultracentri- activity of GSH transferase toward 0.05 to 1.0 mw CDNB or DCNB was fuge. The final supernatant was used as the cytosol fraction. measured in the presence of nafenopin in the assay system. The GSH

CANCER RESEARCH VOL. 45 OCTOBER 1985 5012

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. EFFECTS ON GSH-REQUIRING ENZYMES BY NAFENOPIN concentration for each assay was fixed as for the in vivo kinetics studies. Effects on Liver Catalase Activity. Both nafenopin and phÃ©n Histochemical Examinations. Liver slices taken from each lobe in a obarbital increased catalase activity to a similar level after 6 manner described previously (45) were fixed with ice-cold 95% ethanol weeks of feeding (Table 2). The activity did not increase further followed by embedding in soft paraffin for the GGT reaction or fixed with by 24 weeks of exposure. Histochemical reaction for catalase 10% phosphate-buffered formalin for catalase reaction. Liver tissue revealed reaction-positive granules which represent peroxisomes blocks fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH in the cytoplasms of hepatocytes in control rats (Fig. 1/1). No 7.4, were also prepared for electron microscopy. To demonstrate the peroxidatic activity of catalase in peroxisomes formalin-fixed sections increase in peroxisomes was evident in the hepatocytes of rats were washed with distilled water and incubated with the alkaline 3,3'- fed phÃ©nobarbitalfor 6 or 24 weeks (Fig. 1B) in comparison to diaminobenzidine medium for 2 h (46). Deparaffinized sections were the density in corresponding untreated hepatocytes. In the rats reacted for GGT using L-7-glutamyl-4-methoxy-|3-naphthylamide (VEGA fed nafenopin for 6 or 24 weeks the cytoplasms of hepatocytes Biochemicals, Tucson, AZ) as a substrate (47). were filled with reaction-positive granules (Fig. 1C), indicating peroxisome proliferation. This was confirmed by electron micros copy (Fig. 1D). RESULTS Effects on Rat Liver Cytosolic GSH Transferase Activities. At 6 weeks of feeding of nafenopin the activities of GSH trans- Male rats were fed equimolar concentrations of nafenopin or ferases with CDNB and DCNB as substrates were decreased to phÃ©nobarbitalfor both 6 and 24 weeks. around 41 and 25%, respectively, of the activity of control liver Effects on Body and Liver Weights. In rats fed nafenopin for Table 3). The activity toward ENPP was only slightly reduced. 6 or 24 weeks body weight gain was inhibited and liver weight At 24 weeks the activities toward CDNB and DCNB in nafenopin- was markedly increased (Table 1). PhÃ©nobarbitalincreased liver fed rats were reduced to the same extent as at 6 weeks, while weights at 24 weeks but to a lesser degree than did nafenopin. activity toward ENPP was comparable to that of untreated rats. The relative liver weight expressed as g liver/100 g body weight The feeding of phÃ©nobarbitalfor 6 weeks enhanced the activ increased substantially in the rats fed nafenopin for 6 weeks and ities of GSH transferases towared all three substrates (Table 3). was increased in the rats fed nafenopin or phÃ©nobarbitalfor 24 The activities toward CDNB and ENPP increased further in the weeks. The food intakes measured for 3 weeks from 12 to 14 rats fed phÃ©nobarbitalfor 24 weeks. weeks were not significantly different between untreated, nafen opin- and phenobarbital-fed rats, i.e., 17.1 Â±1.7, 17.1 Â±1.3, Kinetics of Rat Liver Cytosolic GSH Transferases. In the GSH transferase assay when CDNB and DCNB were used as and 17.2 Â±1.8 g/day/rat, respectively. substrates at different concentrations and the GSH concentra Microscopically enlargement of centrilobular hepatocytes was tion was held constant the apparent kinetics of liver cytosolic observed in the rats fed phÃ©nobarbital, whereas nafenopin in GSH transferases in untreated and nafenopin-fed rats at 24 duced a diffuse enlargement of hepatocytes throughout the weeks of feeding appeared to fit Michaelis-Menten kinetics as lobule. Disorders in lobular architecture or formation of abnormal shown in Chart 1, A and B. hepatocellular lesions were not observed in the livers of the rats For GSH transferase activity toward CDNB, the Km for the fed nafenopin or phÃ©nobarbitaleven for 24 weeks. enzyme in nafenopin-fed rats was increased from 0.09 Â±0.01 HIM in controls to 0.13 Â± 0.02 (P < 0.025). The Vmaxwas Table 1 decreased from 590 Â±40 units/mg cytosolic protein to 220 Â± Effects of dietary nafenopinand phÃ©nobarbitalonbody and liver weights 20 (P < 0.001). The Kmof GSH transferase activity toward DCNB Duration6 wt361 wt14.5 g bodywt4.0 were 0.45 Â±0.05 in untreated rats and 0.90 Â±0.12 mw in nafenopin-fed rats (P < 0.005). The Vmaxof GSH transferase weeksExposureControl Â±12" Â±2.2 Â±0.6 Nafenopin 319 Â±14" 20.3 Â±1.5 6.4 Â±0.3Â° activity toward DCNB in untreated and nafenopin-fed rats were PhÃ©nobarbitalBody379 Â±10Liver16.9 Â±1.7gliverwt/1004.5 Â±0.5 39 Â±2 and 24 + 3 units/mg cytosolic protein (P < 0.001), 24 weeks Control 422 Â±9 12.7 Â±0.26 3.0 + 0.1 respectively. Thus the dietary treatment with nafenopin inhibited Nafenopin 331 Â±15 24.0Â±1.4C 7.3 Â±0.1C PhÃ©nobarbital 434 Â±21 18.3 Â±1.5Â° 4.2Â±0.2C GSH transferase activities toward CDNB and DCNB in a similar manner. " Mean Â±SDof 4 rats/group (6 weeks) and 6 rats/group (24 weeks). 6P< 0.005. In Vitro Effects on the Activities of Liver Cytosolic GSH CP< 0.001. Transferases and Their Kinetics. Nafenopin was added to normal rat liver cytosol fractions and the reaction was started Table 2 immediately with addition of CDNB and GSH. At 1.0 and 3.0 mM Effects of dietary nafenopinand phÃ©nobarbitalonliver catalaseactivity in the assay system nafenopin inhibited the GSH transferase activity activity relative to that of corresponding cytosol fractions without (units/mg ExposureControl protein)332 nafenopin (Chart 2A). Maximum inhibition at 3.0 mM was about Â±11a 60%. The GSH transferase activity toward DCNB was also weeksCatalase Nafenopin 555 Â±33" immediately inhibited by addition of nafenopin to the reaction 475 Â±53Â° PhÃ©nobarbitalDuration6 mixture over a range of 0.03 to 3.0 mM (Chart 2ÃŸ).In this case Control 24 weeks 299 Â±65 maximum inhibition at 3.0 mM was about 90%. Nafenopin 464 Â±81" Alterations in kinetics of GSH transferases in the normal liver 508 Â±75" PhÃ©nobarbital cytosol fractions by addition of nafenopin to the assay systems 8 Mean Â±SDof 4 rats/group (6 weeks) and 6 rats/group (24 weeks). 6 P < 0.001. are illustrated in Chart 3, A and B. In the case of GSH transferase CP<0.01. activity with CDNB as a substrate nafenopin appeared to inhibit " P < 0.005. the activity in a competitive type manner (Chart 3A) which

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Table 3 Effects of dietary nafenopinand phÃ©nobarbitalonliver cytosolic glutathione transteraseactivities protein)ExposureControlNafenopin GSH transferase activities (units/mg ,2-Epoxy-3-

weeks 478 Â±102 Â±2.5 Â±2.4 Nafenopin1-Chloro-1,2- 197 Â±35" 7.9 Â±1.1* 28.6 Â±4.0 1109 Â±18163,4-Dichloronitro-49.8 Â±7.361 71.1 Â±16.46 8 Mean Â±SDof 4 rats/group (6 weeks) or 6 rats/group (24 weeks). " P < 0.001. 0P < 0.005.

S Chart 1. Lineweaver-Burk plots of kinetics S. 0.20 of livercytosolic GSHtransferases in nafenopin- O) fed (A) and untreated control (â€¢)ratsat 24 weeks of feeding. The GSH transferase activi ties when CDNB (A) and DCNB (B) were used as variable substrates and GSH concentration was heldconstant were measuredas described I in "Materials and Methods." The cytosolic pro H">o.ooÂ¡ 0.10 tein contents were fixed at 20 /jg/ml and 200 i

levels of these activities did not change further with feeding for 24 weeks. GSH peroxidase activity toward fert-butyl hydroper IM oxide was measured at 24 weeks of feeding and was decreased in the rats fed nafenopin to about 50% of control activity. Feeding of phÃ©nobarbitalto rats for 6 weeks slightly lowered GSH peroxidase activity toward eumene hydroperoxide (Table M 4). In the rats fed phÃ©nobarbitalfor 24 weeks GSH peroxidase activity was comparable to that of untreated rats. In Vitro Effects on Activity of GSH Peroxidase. When nafen opin was mixed with normal rat liver cytosol fractions at 0.01 to 3.0 mW in the standard GSH peroxidase assay system only the 0.01 O.I 0.3 1 3 0.01 O.I 0.3 1 3 highest concentration slightly lowered activity toward H202 rela Chart 2. In vitro effect of nafenopinon normalrat liver cytosolic GSHtransferase tive to that of the control fractions (Chart 4A). In the case of the activities toward CDNB and DCNB. Nafenopin was mixed with normal rat liver cytosols (20 ^g/ml cytosolic protein/ml assay medium)at 0.01 to 3.0 mu (abscissa) activity toward eumene hydroperoxide the activity relative to that in the assay system and the GSH transferase activity was measured immediately of the unexposed fractions was also lowered only when 3.0 mw against CDNB (A) or DCNB (ÃŸ).Activity relative to the corresponding cytosolic activities without nafenopinis represented as percentages (ordinate).Points, mean nafenopin were mixed in the assay system (Chart 4B). of duplicate determinations for 3 rats; bars, SD, and small deviations are not Effects on GGT Activity. In untreated rats liver GGT activity illustrated; *, P < 0.05. was increased 10-fold by 24 weeks of study as compared with the activity at 6 weeks (Table 5). Fedding of phÃ©nobarbital differed from mode of inhibition on the activity toward this enhanced liver GGT activity at both 6 and 24 weeks. In contrast substrate in nafenopin-fed rats (Chart i A). When DCNB was administration of nafenopin lowered the GGT activity at 24 used as the substrate nafenopin inhibited the activity in a non- weeks. competitive manner (Chart 3B) which also was different from the In untreated rats at 6 weeks of study only biliary epithelial cells type of inhibition found in the nafenopin-fed rats (Chart 18) as were histochemically positive for GGT activity. In the rats fed well as that of the in vitro inhibition by nafenopin of the activity phÃ©nobarbital for 6 weeks biliary epithelial cells and a small toward CDNB (Chart 3A). number of hepatocytes around some portal areas displayed Effects on Rat Liver Cytosolic GSH Peroxidase Activities. histochemical GGT activity. At 24 weeks of study in untreated In rats fed nafenopin for 6 weeks the activities of GSH peroxidase rats one to three cell layers of hepatocytes surrounding the with H2O2 and eumene hydroperoxide as substrates were de portal triads displayed GGT activity (Fig. 2A). By 24 weeks of creased to around half the levels of untreated rats (Table 4). The feeding of phÃ©nobarbitallarge periportal areas of GGT-positive

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Chart 3. Lineweaver-Burk plots showing in vitro effects of nafenopin on kinetics of normal 0.020 rat liver cytosolic GSH transferases. The GSH I 0.20 transferase activities when CDNB or DCNB was used as variable substrates and GSH concen 9 tration was held constant were measured as C described in "Materials and Methods." Activity toward CDNB (A) was measured in the pres I ence of nafenopin at 0 (â€¢),1.0(O), and 2.0 (A) o 0.010 mw in the assay system with the use of 20 Â¡Â¿g i 0.10 0.008 cytosolic protein/ml assay medium, while the f^1 40.08 activity toward DCNB (8) was measured in the 0.006 0.08 presence of nafenopin at 0 (â€¢),0.1(O), and 0.2 0.004 0.04 (A) mw in the assay system with the use of 200 B jig cytosolic protein/ml assay medium. Points, 0.002 mean of duplicate determinations for 3 rats. -20 -10 5 10 20 -3-2-1 123*5

Table 4 Effects of dietary natenopin and phÃ©nobarbital on liver cytosolic glutathione peroxidase activity protein)ExposureControl GSH peroxidase activity (units/mg hy- hy- H2026 droperoxide957 droperoxideNot weeks 712 Â±177a Â±44 tested 434 Â±18" 494 Â±40C Nafenopin Not tested 622 Â±134"845 PhÃ©nobarbitalControl Â±8524 518 Nottested311

weeks 734 Â±80 Â±83 Â±15 380 Â±48" 446 Â±40C 147 Â±7Â° Nafenopin PhÃ©nobarbitalDuration 745 Â±51Cumene 973 Â±154fert-Butyl 319 Â±11 " Mean Â±SDof 4 rats/group (6 weeks) or 6 rats/group (24 weeks). bP<0.01. CP< 0.001.

Table 5 X Effects of dietary nafenopin and phÃ©nobarbital on liver y-glutamyltranspeptidase IN activity activity Exposure*Control (units/mgprotein)0.3 Â±0.2a weeks24 Nafenopin 0.5 Â±0.4 1.0Â±0.4"2.6 PhÃ©nobarbitalControl Â±1.2Â° weeksGGT 0.8 Â±0.3" Nafenopin 8.6 Â±3.8" PhÃ©nobarbitalDuration6 8 Mean Â±SD of 4 rats/group (6 weeks) and 6 rats/group (24 weeks). 6P< 0.025. 0.01 0.1 0.3 I 3 â€žM 0.01 0.1 0.3 1 3, c P < 0.01, as compared with rats fed the same diet for 6 weeks. Chart 4. In vitro effect of nafenopin on normal rat liver cytosolic GSH peroxidase dP<0.01. activities. Nafenopin was mixed with normal rat liver cytosols (20 Â»gcytosolic "P< 0.005. P < 0.005, as compared with rats fed the same diet for 6 weeks. protein/ml assay medium) at 0.01 to 3.0 mw (abscissa) in the assay system and the GSH peroxidase activity toward H?02 (A) or eumene hydroperoxide (8) was measured immediately after the 10-min incubation with GSSG reducÃase and NADPH. Activity relative to the corresponding cytosolic activities without nafenopin eration and enhancement of catalase activity. In contrast phÃ©n is represented as percentages (ordinate). Points, mean of duplicate determinations for 3 rats; bars, SD, and small deviations are not illustrated; *, P < 0.05. obarbital enhanced GSH transferase and GGT activities, did not affect GSH peroxidase, and increased catalase activity in the absence of peroxisome proliferation. hepatocytes were present (Fig. 2B). In contrast in the rats fed Prolonged exposure to nafenopin produced a decrease in GSH nafenopin for 24 weeks GGT activity was found only in biliary transferase activity, as has been reported for clofibrate (29) and epithelial cells (Fig. 2C). ciprofibrate (21). The decrease in activity toward DCNB was greater than that toward CDNB and the activity toward ENPP DISCUSSION was not altered. The experiments to examine the in vitro effects of nafenopin on untreated rat liver cytosolic GSH transferase The present study documented marked differences between activities also revealed a greater inhibition of the activity toward the effects of the experimental hepatocarcinogens nafenopin and DCNB than toward CDNB. Nafenopin inhibited in vitro the activity phÃ©nobarbital on liver GSH-requiring enzymes. Nafenopin pro toward CDNB in a competitive manner and that toward DCNB duced a marked suppression of GSH transferase, GSH peroxi in a noncompetitive manner. The latter finding was similar to that dase, and GGT activities in the presence of peroxisome prolif- of Awasthi ef al. (21) who also provided evidence that inhibition

CANCER RESEARCH VOL. 45 OCTOBER 1985 5015 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. EFFECTS ON GSH-REQUIRING ENZYMES BY NAFENOPIN was due to binding of nafenopin to the enzyme subunits. activity in liver altered foci and neoplasms induced in rats by the The in vitro effects of nafenopin, however, may not fully carcinogen W-2-fluorenylacetamide (32). This inhibition of GGT account for the decreases in the GSH transferase activities found activity was suggested to contribute to the reported absence of in cytosols of nafenopin-fed rats. The activity toward CDNB or GGT in liver preneoplastic lesions and hepatocellular carcinomas DCNB was inhibited in nafenopin-fed rats in a mode different in rats exposed to hypolipidemic agents (52-55). In the present from that of in vitro inhibition by nafenopin. It could be that study nafenopin suppressed the age-related appearance of GGT nafenopin metabolites or generation of H2O2 in vivo contribute activity in periportal hepatocytes (43, 56, 57), in contrast to an to enzyme inhibition. Fucci ef al. (48) have reported that inacti- enhancement by phÃ©nobarbital.The appearance of GGT activity vation of enzymes by mixed-function oxidation systems can in adult rat hepatoctes in primary cultures has also been delayed occur through the generation of H2O2 and its conversion to an by addition of nafenopin (58). GGT is an enzyme involved in the activated oxygen species which reacts with the enzymes. mercapturic acid pathway (26, 38) as well as the vglutamyl Feeding of nafenopin decreased GSH peroxidase activity to cycle (59). Therefore the suppression of hepatocellular GGT ward H2O2 and two organic hydroperoxides to around 50% of activity by nafenopin may be an important part of its overall the levels of corresponding controls. In vitro inhibitions by nafen effects on GSH-requiring enzymes. opin of the activities toward H202 and eumene hydroperoxide The present study showed that phÃ©nobarbital,an experimental were also demonstrated. Although the in vitro inhibitions were hepatocarcinogen (33, 34) and liver neoplasm promoter (35, 36), not marked under the conditions used in the present experi exerted biochemical effects quite different from those of nafen ments, in current studies we have found that inhibition is more opin on conjugation and scavenger enzymes requiring GSH. profound if the concentration of H2O2 is decreased (49). H2O2 is PhÃ©nobarbitalat the dose used in this study strongly promotes a substrate specific for selenium-dependent GSH peroxidase liver neoplasm development in rats exposed previously to DNA- (30,50) and 33% of total GSH peroxidase activity toward eumene reactive hepatocarcinogens, whereas studies on the modifying hydroperoxide in rat liver cytosol depends upon selenium-inde effects of peroxisome proliferators on rat hepatocarcinogenesis pendent GSH peroxidase (51). Therefore feeding of nafenopin have not reached a consistent conclusion (32, 37, 60-62). In appears to affect both selenium-dependent and selenium-inde consideration of the differences in the biochemical effects be pendent GSH peroxidases. The selenium-independent GSH per tween phÃ©nobarbitaland nafenopin documented in this study, it oxidase has been shown to be an activity of GSH transferase seems probable that the action of nafenopin in liver carcinogen- (27, 28, 30, 31) but the inhibition of the selenium-dependent esis is quite different from that of phÃ©nobarbital. activity demonstrates that inhibition is not attributable solely to effects on GSH transferases. ACKNOWLEDGMENTS Ciriolo et al. (20) have reported that the activity of GSH peroxidase toward fert-butyl hydroperoxide in rat liver was sup The authors are indebted to Dr. Yohichi Mochizuki for the electron microscopic examinations and for valuable suggestions and to Drs. Edward Butler and Tomiko pressed by administration of clofibrate or fenofibrate for 30 days Shimada for helpful advice. The authors also thank Laura Karabaic and Arieen and that addition of these hypolipidemic drugs to the assay Weinstein for typing the manuscript. system was not effective in inhibiting the activity. On the other hand Awasthi ef al. (21) found only a minimal inhibition of GSH REFERENCES peroxidase activity (substrate not specified) by ciprofibrate in rats fed the agent for 22 days or with in vitro addition. The latter 1. Hess, R., Staubli, W., and Riess, W. Nature of the hepatomegalic effect produced by ethyl-chlorophenoxyisobutyrate in the rat. Nature (Lond.), 208: authors attributed this to their finding that ciprofibrate had the 856-888,1965. lowest affinity for the GSH transferase subunit Yc which ex 2. Staubli, W., Schweizer, W., Suter, J., and Weibel, E. R. The proliferative presses the maximum GSH peroxidase activity. It could be that response of hepatic peroxisomes of neonatal rats in treatment with Su-13437 (nafenopin). J. 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Fig. 1. Hepatic peroxisomes detected by catalase histochemistry and electron microscopy. A, control rat liver at 24 weeks. Reaction-positive granules which represent peroxisomes are present in the cytoplasms of hepatocytes. x 330. B, phenobarbital-treated rat liver at 24 weeks. No apparent increase in peroxisome number is noted as compared with that of corresponding untreated hepatocytes shown in A. x 330. C, nafenopin-treated rat liver at 24 weeks. Hepatocytes are filled with confluent granules indicating peroxisome proliferation, x 330. D, electron micrograph of a hepatocyte from a rat fed nafenopin for 24 weeks. Many large peroxisomes are present (arrows). Several peroxisomes contain crystalloid nucleoids or show flocculent matrices, x 20,000.

CANCER RESEARCH VOL. 45 OCTOBER 1985 5018

fe -V-' * >, ' *â€¢ -tt *Â«*â€¢..

â€¢vvÃ§ â€¢*â€¢ k A f

Fig. 2. Effects of nafenopin and phÃ©nobarbitalon liver GGT activity detected by histochemical reaction. A. control rat liver at 24 weeks. A few hepatocytes surrounding the portal areas and biliary duct epithelial cells have GGT activity. B, phenobarbital-treated rat liver at 24 weeks. Many periportal hepatocytes display GGT activity. C, nafenopin-treated rat liver at 24 weeks. GGT activity is absent in hepatocytes but is still present in biliary duct epithelial cells. All x 22.

CANCER RESEARCH VOL. 45 OCTOBER 1985 5019 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. Effects of the Hepatocarcinogen Nafenopin, a Peroxisome Proliferator, on the Activities of Rat Liver Glutathione-requiring Enzymes and Catalase in Comparison to the Action of Phenobarbital

Kazunori Furukawa, Satoshi Numoto, Keizo Furuya, et al.

Cancer Res 1985;45:5011-5019.

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