Absence of a promoting or sequential syncarcinogenic effect in rat liver by the carcinogenic hypolipidemic drug nafenopin given after N-2-fluorenylacetamide

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

76-85

77,

(1985)

Absence of a Promoting or Sequential Syncarcinogenic Effect in Rat Liver by the Carcinogenic Hypolipidemic Drug Nafenopin Given after A/-2-Fluorenylacetamide SATOSHI NUMOTO,’ HIDERI MORI,~ KEIZO FLJRUYA,~ WALTER G. LEVINE,~ AND GARY M. WILLIAMS’ Naylor

Dana

Institute

for Disease

Prevention,

Received

April

American

Health

28, 1984; accepted

Foundation,

August

Valhalla,

New

York

10595

15, 1984

Absence of a Promoting or Sequential Syncarcinogenic Effect in the Rat Liver by the Carcinogenic Hypolipidemic Drug Nafenopin Given after N-2Fluorenylacetamide. NUMOTO, S., MORI, H., FURUYA, K., LEVINE, W. G., AND WILLIAMS, G. M. (1985). Toxicol. Appl. Pharmacol. 77, 76-85. The hypolipidemic agent nafenopin, (NF), has been reported to be carcinogenic to rat liver. To determine whether nafenopin exerts a promoting or syncarcinogenic effect in rat liver, its effect on liver carcinogenesis induced by N-2-fluorenylacetamide (FAA) was studied. In two separate experiments, male F344 rats were fed 0.02% FAA for either 10 or 8 weeks to induce preneoplastic liver lesions. Following a recovery period of I week, rats were given 0.0 1 or 0.02% NF in the diet for 23 weeks in one experiment and 0.05 or 0.1% for 24 weeks in the other. The final incidence of neoplasms, and their numbers, size distribution, and degrees of differentiation were not significantly different in groups given NF after FAA compared to those maintained on a basal diet after FAA. In the group treated with the highest dose level of NF following FAA, however, there was a decrease in the number of grossly visible small neoplasms. In contrast, the liver neoplasm promoter phenobarbital increased the multiplicity, although not the incidence, of liver neoplasms when given after FAA. Thus, four different dose levels of NF showed no promoting or syncarcinogenic effect on FAA-induced hepatocarcino&Y&S.

0 1985 Academic

Press. Inc

Nafenopin (2-methyl-2-[4-( 1,2,3,4,-tetrahydro- 1-naphthyl)phenoxy] propanoic acid; Su13,437) (NF) is an aryloxyisobutyrate derivative which was developed as a hypolipidemic agent. It induces marked hypertrophy and hyperplasia (Best and Duncan, 1970; Beckett

1972; Reddy et al., 1973, 1974; Leighton et al., 1975) hepatic peroxisome proliferation (Reddy et al., 1974; Leighton et al., 1975; Stiubli et al., 1977) and an increase in the activities of some peroxisome-associated enzymes (Reddy et al., 1974; Moody and Reddy, 1974, 1978; Inestrosa et al., 1979). Feeding of 0.1% (w/w) NF produced hepatocellular carcinomas in 100% of male and female acatalasemic (Csb) mice between 18 and 20 months of the experiment (Reddy et al., 1976) and in 70% of male F344 rats exposed for over 18 months (Reddy and Rao, 1977). This result is similar to the effect of the related drug clofibrate (Reddy and Quret al.,

’ Visiting Scientist from the Second Department of Pathology, Tokushima University School of Medicine, Tokushima, Japan. 2 Present address: Department of Pathology, Gifu University School of Medicine, Gifu, Japan. 3 Present address: Department of Pathology, Ehime Prefectural Central Hospital, Ehime, Japan. 4 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, N.Y. 1046 I. 5 To whom correspondence should be addressed. 0041-008X/85

$3.00

Copyrigt 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved

76

NAFENOPIN

EFFECTS ON LIVER CARCINOGENESIS

eshi, 1979; Svoboda and Azamoff, 1979; Blane and Pinaroli, 1980) and gemfibrozil (Fitzgerald et al., 1981). The mechanism by which such peroxisome-proliferating agents exert their hepatocarcinogenic effects is not known, although they have been postulated by Reddy et al. (1980) to constitute a special class of carcinogens. Chemical carcinogens have been categorized as genotoxic or epigenetic according to whether they damage DNA or exert another biologic effect that could account for their carcinogenicity (Weisburger and Williams, 1980; Williams, 1983). NF has not been found to be genotoxic (Warren et al., 1980; Williams, 198 1b), nor have other peroxisomeproliferating agents (Warren et al., 1980; Blane and Pinaroli, 1980; Fitzgerald et al., 198 1; Albro et al. 1983; Von Daniken et al., 1984; Linnainmaa, 1984; Williams et al., 1984); these agents, therefore, are candidate epigenetic carcinogens. One class of epigenetic or secondary carcinogens is neoplasm promoters (Weisburger and Williams, 1980). These are considered to be carcinogenic by virtue of a promoting effect on preexisting neoplastic cells. In particular, a number of liver carcinogens such as phenobarbital (PB) and DDT have been proposed to be carcinogenic on this basis (Williams, 1978, 198 la). Three other agents that, like NF, produce peroxisome proliferation, Wy-14,643, clofibrate, and di(2-ethylhexyl) phthalate, have been reported to exert an enhancing effect on liver carcinogenesis when given after diethylnitrosamine (Mochizuki et al., 1982; Reddy and Rao, 1978; Ward et al., 1983). Although suggestive of a promoting action, such observations are not proof, since they could also result from a syncarcinogenic effect of two carcinogens given in sequence (Williams, 1984; Williams and Furuya, 1984). NF has also been suggested by Schulte-Hermann et al. (198 1) to be a promoter based on its stimulatory effect on DNA synthesis in liver altered foci induced by N-nitrosomorpholine. Therefore, to elucidate the mechanism of action of NF, we

77

undertook a study to determine if NF would enhance liver carcinogenesis when given after a genotoxic liver carcinogen. In a somewhat similar study, Staubli et al. (1984) reported that, in contrast to the enhancing effects reported for other peroxisome-proliferating agents, NF inhibited the formation of liveraltered foci induced by diethylnitrosamine. To study the effect of NF on liver carcinogenesis, we used a model system in which rats are exposed to N-2-fluorenylacetamide (FAA) to induce small foci of altered liver cells following which the test chemical is administered to determine its effect on the foci and the incidence of early liver neoplasms (Williams, 1982). This model has the advantage that carcinogen-induced “initiated” cells are present before exposure to the test enhancing agent is commenced. Thus, cocarcinogenic effects on the stage of neoplastic conversion are largely precluded. In this system, the enhancing effect on hepatocarcinogenesis has been demonstrated for the drugs PB (Watanabe and Williams, 1978) barbital (Mori et al., 1981) and methapyrilene (Furuya et al., 1983) while a series of benzodiazepines have been inactive (Remandet et al., 1984). This protocol also permits the detection of any syncarcinogenic effect of the second agent (Williams et al., 1981; Furuya and Williams, 1984) which could result from DNA reactivity or other summative genotoxic effects (Williams, 1984). In this paper, we report that NF did not enhance the hepatocarcinogenicity of previously administered FAA and, therefore, did not exert either a promoting or syncarcinogenie effect. METHODS Male F344 rats were obtained from Charles River Laboratory, Kingston, New York, were kept in groups of three in solid-bottom polycarbonate cages with hardwood bedding, and were housed in a conventionally maintained animal facility maintained at 20 + 2°C and 50 f 10% relative humidity with a 12-hr light-dark cycle. Body weight was measured once every 4 weeks. Two independent experiments were conducted.

78

NUMOTO

Experiment 1. Sixty-nine rats were divided into four groups. Starting at 8 weeks of age, groups I-I, 1-2, and 1-3 were fed 0.02% FAA (Aldrich Chemical Co., Milwaukee, Wise.) in a basal diet (NIH-07) for 10 weeks. After this initiation period, FAA administration was discontinued and the animals were fed the basal diet for 1 week. Then, the rats in each group were given the basal diet with no addition (group l-l) or containing either 0.01% (group l-2) or 0.02% (group l-3) NF (CIBA Pharmaceutical Co., Summit, N.J.) for 23 weeks. The rats in group l-4 were fed the basal diet for the first 11 weeks and the basal diet plus 0.02% NF for the following 23 weeks. All animals were killed at Experiment Week 34. Experiment 2. One hundred two rats were divided into seven groups. Group 2-1 was maintained as the untreated control. Starting at 8 weeks of age, groups 2-2, 2-3, 2-4, and 2-5 were fed 0.02% FAA in a basal diet for the first 8 weeks. After this initiation period, FAA administration was discontinued and the animals were fed the basal diet for 1 week. Then, each group was given the basal diet with no addition (group 2-2) or either containing 0.05% NF (group 2-3), 0.1% NF (group 2-4) or 0.05% PB (Mallinckrodt Chemical Works, St. Louis, MO.) (group 2-5) for 24 weeks. Groups 2-6 and 2-7 were fed the basal diet for the first 9 weeks and then given 0.1% NF or 0.05% PB in the basal diet for 24 weeks, respectively. All the animals were killed at Experimental Week 33. Macroscopic and histologic observation. Animals were killed in a carbon dioxide chamber. The whole livers were removed and grossly visible tumors on the surface were measured and counted. After that, liver slices, taken in a standard manner from each lobe (Williams and Watanabe, 1978) were fixed in 10% phosphate-buffered formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin-eosin for histological examination. The neoplastic nodules and hepatocellular carcinomas were quantified by the histologic criteria of the Institute of Laboratory Animal Resources monograph (Stewart et al., 1980).

RESULTS Two feeding studies were conducted, one in which rats were fed 0.01 or 0.02% NF in the diet after FAA and a second in which doses of 0.05 or 0.1% were given after FAA (Table 1). Effects of Treatments Weights

on Body and Liver

Feeding of NF depressed the body weight gain of rats. In Experiment 1, the body

ET AL.

weights in group l-3 given 0.02% NF for 23 weeks following FAA were significantly lower than those in group l-l treated with FAA alone (Table 1). In Experiment 2, higher doses (0.05 and 0.1%) of NF seemed to have a greater depressing effect on body weight gain than the lowe; doses (0.01 and 0.02%) used in Experiment 1. The body weights in groups 2-3, 2-4, and 2-6 treated with NF were significantly different from the appropriate control groups. In contrast, PB (groups 2-5 and 2-7) had a tendency to increase the body weight gain. The absolute and relative liver weights (Table 1) in all groups treated with NF were remarkably higher than those in appropriate comparison groups. The effect of NF alone was documented in group 2-6 given NF after administration of only the basal diet; the average liver weight in this group was 22.4 g compared to 13.9 g in group 2- 1 maintained on basal diet throughout the experiment. PB also increased these parameters. The highest absolute and relative liver weights were observed in group 2-4 treated with highest dose (0.1%) of NF following FAA. Effects of Treatments on Liver Pathology In Experiment 1, all of the livers in rats treated with FAA (groups l- 1, l-2, and l-3) had many yellowish-white or gray nodular lesions. Some of the large lesions which were elevated above the surface level of the liver contained portions with hemorrhage or necrosis. The average numbers and size distribution of grossly visible tumors are summarized in Table 2. The great majority of tumors were less than 3 mm in diameter. There were no significant differences between the average numbers of tumors in groups treated with NF after FAA (groups l-2 and l-3) and that group given FAA alone (group l-l). The incidence of histologically confirmed liver neoplasms is shown in Table 3. Administration of FAA whether or not followed by NF (groups l-1, 1-2, and 1-3) induced neoplastic nodules in all the animals and carci-

NAFENOPIN

TABLE EFFECT

79

EFFECTS ON LIVER CARCINOGENESIS 1

0F DIETARY ADMINISTRATION 01= iV-2-~UORENYLA~ETAMIDE, AND PHENOBARBITAL ON LIVER AND BODY WEIGH’B

NAFENOPIN, OF RATS

Exposure b Group

First

Second

Expt 1 l-l l-2 l-3 l-4

FAA (10) FAA (10) FAA (10) Basal (10)

Basal (23) 0.01% NF (23) 0.02% NF (23) 0.02% NF (23)

18 18 17 15

17.6 f 20.2 * 22.0 f 16.0 f

2.2“ 3.1’ 5.8c 1.2

377 373 349 381

f f + f

30 29 33d 29

4.7 5.4 6.5 4.2

+ f f rt

0.7 0.9d 2.5’ 0.2

Expt 2 2-1 2-2 2-3 2-4 2-5 2-6 2-7

None FAA FAA FAA FAA None None

None (24) None (24) 0.05% NF (24) 0.1% NF (24) 0.05% PB (24) 0.1% NF (24) 0.05% PB (24)

12 14 15 15 14 14 15

13.9 15.7 20.5 22.5 18.0 22.4 18.5

1.5 2.7 2.3’ 1.8e 2. I J 1.9e 1.6’

356 349 305 315 360 319 380

+ 22 + 18 + 13c + 16’ -c 17 + 15e + 23d

3.9 4.5 6.7 7.1 5.0 7.0 4.9

f f f k + f f

0.4 0.6 0.7’ 0.5’ 0.4d 0.4’ o.4c

(8) (8) (8) (8) (8) (8) (8)

Liver wt (8)

+ + iz k + f +

Body wt (g)

Liver wt/ MYW V-J)

No. of rats

a Values are X + SD. bAll exposures were separated by an interval of 1 week on the basal diet. Numbers in parentheses indicate number of weeks exposure continued. ‘-‘Significantly different from appropriate control group by Student’s 1 test, ‘p < 0.01, “p < 0.025, ‘p < 0.001, Jp< 0.05.

in half or more of them. There were no differences between the degree of neoplasm differentiation, numbers and incidences of neoplastic nodules, and carcinomas in these groups. Neoplastic nodules were usually well circumscribed, compressed the plates and sinusoids of the surrounding liver tissue, and showed architectural distortions. The cells in

most of the neoplastic nodules had large foamy and acidophilic cytoplasms and hyperchromatic nuclei with prominent nucleoli. A minority of the neoplastic nodules were either basophilic or contained a mixture of acidophilic and clear cells. Mitotic figures were often numerous and abnormal. Most hepatocellular carcinomas displayed a typical

nomas

TABLE 2 NUMBER

AND

SIZE DISTRIBUTION OF GROSSLY VISIBLE AFTER N-2-FLUORENYLACETAMIDE

LIVER TUMORS IN EXPERIMENT

IN RATS I

FED NAFENOPIN

No. of tumors relative to size

Exposure” Group

First

Second

No. of rats

1-I 1-2 1-3 1-4

FAA (10) FAA (IO) FAA (10) Basal (10)

Basal (23) 0.01% NF (23) 0.02% NF (23) 0.02% NF (23)

18 18 17 15

<3 mm 83.5 64.4 71.3 0.3

f 37.0” + 39.8 + 38.2 -+ 0.6

3-7 mm

>7 mm

0.6 f 0.8 0.8 + 1.0 0.8 f 0.9 0

0.5 f 0.5 1.1 -c 1.1 0.4 f 0.8 0

“All exposures were separated by an interval of I week; thus, the total experimental period was 34 weeks; numbers in parentheses indicate number of weeks of exposure. b Values are X + SD.

80

NUMOTO

ET AL

TABLE 3 LIVERNEOPLASMSIN

RATSFEDNAFENOPINAFTERN-2-FLUORENYLACETAMIDEIN

Exposure” Group

First

Second

No. of rats

I-1 l-2

FAA (10) FAA (10)

Basal (23) 0.01% NF (23)

18 18

1-4

Basal (10)

0.02% NF (23)

15

EXPERIMENI

1

Average No. of neoplastic nodules per rat

Average No. of hepatocellular carcinomas per rat

13.5 f 6.4h (18) 11.7 f 4.4 (17) 0

0.8 t 0.8 (1 I) 1.1 + 1.4 (9) 0

” All exposures were separated by an interval of I week; thus the total experimental period was 34 weeks. ’ Values are X + SD. Numbers in parentheses indicate numbers of rats with neoplasms.

trabecular pattern and consisted predominantly of basophilic cells. A few had areas with adenomatous patterns. Only one neoplasm in group l-3 (0.02% NF following FAA) was poorly differentiated and metastasized to the lung. In group 1-4, treated with NF alone, no preneoplastic or neoplastic lesions were observed. There were a few small areas of bile duct proliferation. In the second experiment with higher doses of NF, grossly visible tumors were almost the same in appearance as those in Experiment 1, but the numbers and size distribution of the tumors were different, as summarized in

Table 4. The highest dose of NF following FAA (group 2-4) significantly decreased the average number of tumors less than 3 mm in diameter compared to that in group 2-2 treated with FAA alone. PB following FAA (group 2-5) significantly increased the number of tumors less than 7 mm in diameter. The numbers in other groups were not different from those in the appropriate control groups. The incidence of histologically confirmed neoplastic nodules and hepatocellular carcinomas is shown in Table 5. NF given after FAA (groups 2-3 and 2-4) did not significantly increase the incidence of neoplastic nodules. In fact, if anything, the numbers of affected

TABLE 4 NUMBERANDSIZEDISTRIBUTION OFGROSSLY VISIBLE LIVERTUMORSIN ~HEN~BARB~TALAFTERN-~-FL~~REN~LA~ETAMIDEINEXPERIMENT

Exposure”

RATSFEDNAFENOPIN 2

OR

No. of tumors relative to sizeb

Group

First

Second

No. of rats

2-1 2-2 2-3 2-4 2-5 2-6 2-7

None (8) FAA (8) FAA (8) FAA (8) FAA (8) None (8) None (8)

None (24) None (24) 0.05% NF (24) 0.1% NF (24) 0.05% PB (24) 0.1% NF (24) 0.05% PB (24)

12 14 15 15 14 14 15

<3 mm 0.2 +- 0.6 31.3 f 13.3 31.1 f 11.6 16.8 f ll.OC 53.8 f 21.8’ 0.3 f 0.8 0.4 + 1.3

3-7 mm 0.1 0.4 0.1 1.1

0 * 0.3 f 0.6 + 0.3 rt 1.4d 0 0

>7 mm 0 0.2 +- 0.8 0 0 0.5 f 0.8 0 0

’ All exposures were separated by an interval of 1 week; thus, the total experimental period was 33 weeks. b Values are X f SD. c,dSignificantly different from group 2 by Student’s t test, ‘p< 0.025, dp< 0.01.

NAFENOPIN

81

EFFECTS ON LIVER CARCINOGENESIS TABLE 5

LIVERNEOPLASMSINRATSFEDNAFENOPINORPHENOBARBITAL N-2-FLUORENYLACETAMIDE INEXPERIMENT~

Exposure” Group

First

Second

No. of rats

2-1 2-2 2-3 2-4 2-5 2-6 2-7

None (8) FAA (8) FAA (8) FAA (8) FAA (8) None (8) None (8)

None (24) None (24) 0.05% NF (24) 0. I% NF (24) 0.05% PB (24) 0.1% NF (24) 0.05% PB (24)

12 14 15 15 14 14 15

AFTER

Average No. of neoplastic nodules per rat’ 0.6 + 0.1 + 0.1 + 1.5 f

0 1.0 (5) 0.3 (2) 0.3 (2) 1.1’(9) 0 0

Average No. of hepatocellular carcinoma per ratb 0 0.2 f 0.8 (1) 0 0 0.2 + 0.4 (3) 0 0

’ All exposures were separated by an interval of 1 week; thus the total experimental period was 33 weeks. * Values are X f SD. Numbers in parentheses indicate numbers of rats with neoplasms. ’ Significantly different from group 2 by Student’s t test, p < 0.05.

rats, i.e., two in each group, were less than those in group 2-2, five rats. NF alone (group 2-6) did not induce either neoplastic nodules or carcinomas. In contrast, administration of PB after FAA (group 2-5) appeared to increase the incidence of rats with neoplasms, 9/14 rats with nodules and 3/14 with carcinomas versus 5/ 14 and l/ 14, respectively, in group 2-2, but these differences were not significant. Nevertheless, PB significantly increased the number of neoplastic nodules in FAA-initiated rat liver. The appearances of liver neoplasms were similar to those in Experiment 1, although no poorly differentiated carcinomas or metastatic foci were observed. No neoplasms or preneoplastic lesions occurred in control rats or those given NF or PB alone. Microscopically, in the livers of rats given only NF (group 2-6), there was no evidence of focal lesions or bile duct proliferation. DISCUSSION In two separate experiments with four different dose levels, NF produced no enhancement of hepatocarcinogenesis in rats previously fed FAA. To the contrary, at the highest dose of NF tested (0. l%), a decrease in the number of grossly visible tumors less

than 3 mm in diameter was observed. The doses of NF used clearly affected the liver as shown by the induced hepatomegaly. Moreover, in one of these studies, the liver neoplasm promoter PB, given after FAA, produced a substantial increase in liver neoplasms, as in previous studies with this protocol (Watanabe and Williams, 1978), thereby demonstrating that the conditions of the present study were adequate for detection of enhancing effects. In addition to the absence of an enhancing effect of NF, the agent by itself also did not produce neoplasms or even preneoplastic lesions within the duration of the experiment. This finding may be due to the fact that administration was for only 6 months, whereas Reddy and Rao (1977) reported that liver tumors occurred only after 18 months of NF administration. The present observations, therefore, show that under the test conditions used, NF did not exert either a syncarcinogenic or promoting action to augment FAA-induced hepatocarcinogenesis. Syncarcinogenesis is the additive or synergistic effect of two carcinogens given together or sequentially (Schmiihl, 1980). This phenomenon was originally described for carcinogens of the type that are damaging to DNA. Thus, syncarcinogenesis has been suggested to result, at least in part,

82

NUMOTO

from summation of the DNA damage produced by two genotoxic carcinogens (Williams et al., 198 1; Williams, 1984). In a previous study, we showed that under conditions similar to the present, administration after FAA of a totally subcarcinogenic dose of diethylnitrosamine, 10 ppm in the drinking water, produced marked syncarcinogenesis (Williams et al., 1981). Moreover, the antihistamine, methapyrilene, which has not been established to be genotoxic, also was syncarcinogenic with FAA when given either before or after it (Furuya and Williams, 1984). Considering the sensitivity of the system, the absence of a syncarcinogenic effect by NF following FAA requires explanation, since this seems to represent one of the few examples where two carcinogens with the same target organ do not have an additive or synergistic effect. One implication of these observations is that NF is not DNA damaging in vivo. This is consistent with its lack of genotoxicity in in vitro short-term tests (Warren et al., 1980) including the rat hepatocyte primary culture/DNA repair test (Williams, 198 lb). Alternatively, Reddy et al. (1980) have proposed that peroxisome-proliferating agents may indirectly give rise to DNA damage mediated by HzOz generated during increased /3 oxidation of lipids stemming from peroxisome proliferation. This hypothesis has not yet been substantiated, but if it proves to be correct, it would seem that this type of DNA damage is not necessarily additive with that of DNA-reactive liver carcinogens. Another explanation for the lack syncarcinogenesis could be that NF produces other effects that retard liver tumor development. Recently, Staubli et al. (1984) have reported that NF inhibited the development of diethylnitrosamine-induced altered foci believed to be precursors of liver neoplasms. Similarly, Perera and Shinozuka (1984) have demonstrated inhibition by two other peroxisome proliferators, BR93 1 and di(2-ethylhexyl) phthalate. The absence of an enhancing effect by NF was unexpected in light of the reports of

ET

AL.

enhancement of liver carcinogenesis by other hypolipidemic agents, Wy- 14,643 (Reddy and Rao, 1978) and clofibrate (Reddy and Rao, 1978; Mochizuki et al., 1982) and the report by Schulte-Hermann et al. ( 198 1) that NF stimulated thymidine incorporation in altered foci of the liver. Reddy and Rao (1978) reported that both 0.5% clofibrate and 0.1% Wy- 14,643 enhanced hepatocarcinogenesis after diethylnitrosamine initiation in rats, and Mochizuki et al. (1982) reported that 0.1 and 0.25% clofibrate markedly enhanced the development of diethylnitrosamine-induced rat liver tumors. Ward et al. (1983) also found enhancement by di(2-ethylhexyl) phthalate, but in mice. In the present experiments, doses of NF comparable to clofibrate were studied, and, as noted, these were biologically active. The reason for this difference between NF and other peroxisome-proliferating agents is unclear, especially since NF induces a profound hepatic hyperplasia and hypertrophy (Best and Duncan, 1970; Beckett et al., 1972; Reddy, et al., 1973, 1974; Leighton et al., 1975), reflected as increased liver weight in the present study. Moreover, NF did not produce an enhancement, but at the highest dose, it actually reduced the number of liver neoplasms. A similar inhibitory effect of clofibrate at high dose (1%) was suggested by Mochizuki et al. (1982) to be partly due to decreased feed intake. Likewise, a poor nutritional state and/or hepatotoxicity might be the cause for the inhibitory effect of NF at the highest dose in the present study. Regardless, it must be concluded that, under the present experimental conditions, NF did not have a promoting effect on FAA-induced hepatocarcinogenesis in rats. This result is consistent with the observations of Smubli et al. ( 1984) and Perera and Shinozuka (1984) on the lack of enhancing effect on liver foci of several peroxisome proliferators, including NF. The basis for the lack of a promoting effect by NF deserves consideration. Like clofibrate, it is hypolipidemic and leads to proliferation of hepatic peroxisomes (Reddy et al., 1974;

NAFENOPIN

EFFECTS ON LIVER CARCINOGENESIS

Leighton et al., 1975; Stiiubli et al., 1977). Like PB, it produces hepatocellular hyperplasia and hypertrophy (Beckett et al., 1972). However, unlike both of these substances, and most other known hepatotrophic and promoting compounds (Schulte-Hermann and Parzefall, 198 l), NF does not induce the hepatic mixed-function oxidase system to an appreciable extent (Beckett et al., 1972; Levine, 1974). Enzyme induction has not yet been shown to be necessary for promotion in the liver, but remains a possibility. It may also be that NF does not produce the effects on the cell membrane that have been suggested to be relevant to promotion (Williams, 1981a). In fact, we have found that NF inhibits y-glutamyl transpeptidase activity (Numsto et al., 1984), a membrane enzyme induced by liver neoplasm promoters (Furukawa et al., 1984; Remandet et al., 1984). This action, incidentally, may account in part for the observations of Sdubli et al. (1984) and Perera and Shinozuka (1984) on reduction of y-glutamyl transpeptidase-positive foci by peroxisome proliferators. Nevertheless, these findings indicate that the biochemical effects of peroxisome proliferators are not limited to peroxisomes. The absence of a liver neoplasm-promoting effect of NF has implications regarding its mechanism of carcinogenesis. NF has produced liver cancer in both mice (Reddy et al., 1976) and rats (Reddy and Rao, 1977). Considering the limited organotropism and apparent nongenotoxicity of NF, it is possible that NF is a type of epigenetic or secondary carcinogen that exerts its effects through indirect mechanisms not involving interaction with DNA. The hepatocarcinogenicity of certain non-DNA-reactive agents with promoting activity has been suggested to be due to their enhancement of growth of preexisting neoplastic cells (Williams, 1978, 1981a). Based on the present observation of a lack of enhancement of FAA-induced foci, NF does not appear to operate by such an action. Thus, the suggestion by Reddy et al. (1980) that peroxisome-proliferating agents are car-

83

cinogenic through an excess generation of potential DNA-damaging oxygen radicals as a result of sustained elevation of the peroxisomal fatty acid P-oxidation system remains a possible explanation. Such a mechanism is distinct from direct DNA reactivity and implies that NF would be carcinogenic only under conditions leading to sufficient generation of oxygen radicals to alter DNA. From the present observations of a lack of syncarcinogenicity with a 6-month exposure, it would seem that long-term administration, as in the study of Reddy and Rao (1977), is required. REFERENCES ALBRO, P. W., CORBETT, J. T., SCHROEDER,J. L., AND JORDAN, S. T. (1983). Incorporation of radioactivity from labeled di-(2ethylhexyl) phthalate into DNA of rat liver in vivo. Chem.-Biol. Interact. 44, l-16. BECKEM’, R. B., WEISS, R., STITZEL, R. E., AND CENEDELLA, R. J. (1972). Studies on the hepatomegaly caused by the hypolipidemic drugs nafenopin and clofibrate. Toxicol. Appl. Pharmacol. 23, 42-53. BEST, M. M., AND DUNCAN, C. H. (1970). Lipid effects of a phenolic ether (SU-13437) in the rat: Comparison with CPIB. Atherosclerosis 12, 185-192. BLANE, G. F., AND PINAROLI, F. (1980). Fenofibrate: Animal toxicology in relation to side-effects in man. Now. Presse Med. 9, 3737. FITZGERALD, J. E., SANYER, J. L., SCHARDEIN, J. L., LAKE, R. S., MCGUIRE, E. J., AND DE LA IGLESIA, F. A. (1981). Carcinogen bioassay and mutagenicity studies with the hypolipidemic agent gemfibrozil. J. Natl. Cancer Inst. 67, 1105-l 116. FURUKAWA, K., MAEURA, Y., F~JRUKAWA, N. T., AND WILLIAMS, G. M. (1984). Induction of butylated hydroxytoluene of rat liver y-glutamyl transpeptidase activity in comparison to expression in carcinogeninduced altered lesions. Chem.-Biol. Interact. 48, 43-58. FURUYA, K., MORI, H., AND WILLIAMS, G. M. (1983). An enhancing effect of the antihistaminic drug methapyrilene on rat liver carcinogenesis by previously administered N-2-fluorenylacetamide. Toxicol. Appl. Pharmacol. 70,49-56. FURUYA, K., AND WILLIAMS, G. M. (1984). Neoplastic conversion in rat liver by the antihistamine methapyrilene demonstrated by a sequential syncarcinogenic effect with N-2-fluorenylacetamide. Toxicol. Appl. Pharmacol. 74, 63-69. INESTROSA,N. C., BRONFMAN, M., AND LEIGHTON, F.

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