Role of testosterone in the induction of hepatic peroxisome proliferation by clofibrate

Role of Testosterone in the Induction of Hepatic Peroxisome Proliferation by Clofibrate Harbhajan S. Paul, Gail Sekas, and Stephen J. Winters Hepatic peroxisome proliferation is induced by a number of agents, including clofibrate. Sustained proliferation of peroxisomes is associated with the development of hepatocellular carcinoma. In the present study, we have investigated the role of testosterone in peroxisome proliferation induced by clofibrate. Three groups of male rats (intact, castrated, and castrated replaced with testosterone) were studied. Proliferation of peroxisomes was induced by feeding clofibrate (0.25%, 0.50%, and 1.0% of diet) for 2 weeks. Peroxisome proliferation was monitored by measuring total peroxisomal B-oxidation activity. In intact rats, the peroxisomal p-oxidation activity (nmol/min/mg protein) increased in a dose-dependent manner and was 7.2 + 0.4. 52.6 f 7.5, 63.2 f 3.7, and 92.4 2 4.0 at clofibrate doses of 0%. 0.25%. 0.50%, and 1.0%. respectively. In contrast, in castrated rats, the total peroxisomal (J-oxidation activity was significantly (P c .Ol) lower at clofibrate levels of 0.25% and 0.50% (25.8 f 2.7 and 42.5 * 2.2, respectively), but not at the clofibrate level of 1 .O% (85.0 f 6.3). Testosterone replacement of castrated rats restored the peroxisomal @oxidation activity. To determine whether the above results were related to the metabolism of clofibrate in the absence or presence of testosterone, we measured serum clofibrate levels. These levels were 50% lower in castrated rats than in intact rats or in testosterone-treated castrated rats. The activity of hepatic uridine diphosphate (UDP)-glucuronyltransferase, the enzyme catalyzing the glucuronidation of clofibrate, was measured using either bilirubin or 4-methylumbelliferone as substrates and was found to be unaffected by castration or testosterone treatment. We conclude that (1) clofibrate-induced proliferation of peroxisomes is blunted in the absence of testosterone; (2) the sexual dimorphism in the response of hepatic peroxisomes to clofibrate results from higher circulating clofibrate concentration in the presence of testosterone; and (3) the metabolic interaction between testosterone and clofibrate for this effect remains to be investigated. Copyright 0 1994 by W.B. Saunders Company

H

proliferation is induced by EPATIC PEROXISOME a wide variety of chemicals, including phthalate ester plasticizers, industrial solvents, and drugs such as the fibrate class of hypolipidemic agents.’ Long-term administration of peroxisome proliferators results in the development of hepatomegaly and hepatocellular carcinoma in rodents.? The mechanism by which peroxisome proliferators induce hepatic tumors is intriguing because these compounds are not mutagenic and do not bind to and directly damage DNA.? Instead, the carcinogenicity of peroxisome proliferators has been hypothesized to be an indirect effect related to the induction of peroxisomeassociated enzymes.?~~ Hepatic peroxisomes contain more than 40 enzymes, including those capable of producing and degrading HzO;. The induction of individual peroxisomal enzymes during peroxisome proliferation varies markedly. For example, treatment with clofibrate produces a several-fold increase in the activity of H20z-generating fatty acyl-coenzyme A (CoA) oxidase, but a minimal increase in the Hz02-

From the Departments c$ Medicine and Molecular Genetics and 3iochern~st~, University of Pittsburgh School of Medicine, Pittsburgh, PA. Submitted September 24, 1992; accepted Aprioril4, 1993. Supported b_vGrant No. IRG-58-30 from the American Cancer SocieQ (H.S.P.) and by National Institutes of Health Grant No. ROI-HD19546 (S.J.lK). Presented in part at the American Gastroenterological Association Meeting, San Francisco. CA, May 1992, and preLio&y published in abstract form (Gastroenterolog?, 102:A882, 1992). Address reprint requests to Harbhajan S. Paul, PhD, Department of Medicine. Montejiore University Hospital, 3459 Fifih Ave. Pittsbu& PA 15213. Copyright 8 1994 by JKB. Saunders Company 00%049519414302-0008$03.00l0 168

metabolizing enzyme catalase.‘J’ Because of this imbalance between H202-generating and -metabolizing enzymes, hepatic cells are subjected to increased oxidative stress. It has been hypothesized that this stress is related to hcpatocarcinogenesis.’ Hydrogen peroxide generated during peroxisomal proliferation causes DNA damage.x-i” For cxamplc. the levels of &hydroxydeoxyguanosine in liver DNA are significantly increased in rats treated with pcroxisomal proliferators.iO,li The factors involved in the development of hepatocellular carcinoma by peroxisome proliferators are not known. Several previous studies have implicated testosterone in the development of liver tumors in laboratory animals.“-iJ Therefore, in the present study we have investigated the role of testosterone in the hepatic peroxisome proliferation induced by the hypolipidemic drug. clofibrate. MATERIALS

AND METHODS

Materials Clofibrate was obtained from Ayrrst Laboratories (New York. NY). Testosterone, palmitoyl-CoA, 4-methylumbelliferone. bilirubin, uridinediphosphoglucuronate, and other analytical grade reagents were obtained from Sigma Chemical (St Louis, MO). Animals

and Treutment

Male Sprague-Dawley rats (Zivic Miller Laboratories, Allison Park, PA) weighing 102 ? 3 g (mean + SE. n = 72) were used in this study; they were housed in individual cages in air-conditioned quarters with a controlled 12hour light/dark cycle. Three groups of rats, namely intact rats. castrated rats, and castrated rats repleted with testosterone. were studied. Castration was performed via a scrotal incision under metofane anesthesia. and animals were maintained for 10 days to allow regression from the effects of endogenous testosterone before clofibrate treatment. Testosterone was replaced using a 1 x 0.125in OD silastic capsule (Dow Corning, Midland, MI) packed with testosterone and imMetabolism, Vol43, No 2 (February), 1994: pp 168-173

TESTOSTERONE

plantetl

AND PEROXISOME

subcutaneously

at the

169

PROLIFERATION

time

of castration.

Within

each

group, rats were treated with one of four levels (0%. 0.25% 0.50%, and 1.00% of diet) of clofibrate for 2 weeks (six animals per group per clofibrate level). Animals were killed by decapitation.

Table 1. Effect of Clofibrate Treatment on Liver Weight Dietary

Levels

of Cloflbrate

0

Analytical Methods Total peroxisomal P-oxidation activity was measured in the 600 x K supernatant fraction of liver homogenates using the palmitoyl-CoA-dependent, potassium cyanide-insensitive, NAD reduction as described by Lazarow.” Uridine diphosphate (UDP)glucuronyltransferase activity was assayed in liver homogenates using bilirubin as the aglycone as described by Heirwegh et al.“> and using 4-methylumbelliferone as the aglycone as described hy Frei et al.” Serum testosterone levels were measured by radioimmunoassay.‘X Serum clofibrate levels were determined spectrophotometrically as outlined by Wolfe et al.l”Protein concentration was measured by the Lowry method using bovine serum albumin as the standard.?”

0.25

0.50

1.oo

Data were analyzed by two-way ANOVA.” When significant F values were obtained, means were compared with Duncan’s multiple range test.?’ Differences were considered statistically significant at P less than .05. RESULTS

Serum Testosterone Levels Serum testosterone levels were measured in intact, castrated, and testosterone-replaced castrated male rats 24 days after surgery. These animals did not receive clofibrate trcatmcnt. As expected, serum testosterone levels were markedly decreased in castrated male rats compared with intact male rats (32 5 1 v 274 2 26 ng/dL, mean 2 SEM of six rats, P < .Ol). Implantation of testosterone capsules into castrated male rats restored serum testosterone levels to those of intact male rats (240 2 15 ng/dL, mean f SEM of six rats, P = NS). Clofibrate treatment for 14 days did not affect serum testosterone levels of either intact or castrated male rats (results not shown). Liver Weights Clofibrate treatment of intact male rats is known to produce hepatomegaly. IX To determine whether testosterone plays a role in mediating the effect of clofibrate, liver weights were measured. Liver weights are expressed as percent body weight to equalize for possible differences in body weight, and are shown in Table 1. Without clofibrate treatment, there were no significant differences in fiver weight between intact, castrated, and castrated male rats with testosterone replacement. Clofibrate treatment increased liver weight in all groups in a dose-dependent fashion. With cfofibrate treatment at 0.25% and 0.50% of the diet, liver weights were significantly lower in castrated than in intact male rats (Table 1). When castrated male rats repleted with testosterone were treated with clofibrate, a degree of hepatomegaly was observed similar to that in intact male rats (Table 1). When the clofibrate level of the diet was increased to 1.0%. there were no significant differences in liver weight between intact and castrated male rats (Table 1). However, the liver weight of castrated

Group

(% of body weight)

Intact male rats

4.95 + 0.17a

Castrated male rats

4.84 t 0.06a

Castrated + testosterone

4.91 + 0.05a

Intact male rats

6.36 + 0.14b

Castrated male rats

5.10 + 0.098

Castrated + testosterone

6.31 + 0.15b

Intact male rats

7.45 5 0.28cd

Castrated male rats

6.40 + 0.13”

Castrated + testosterone

7.44 -t 0.28Cd

Intact male rats

7.37 * 0.17Cd

Castrated male rats

7.21 * 0.18c

Castrated + testosterone

7.89 2 0.17d

NOTE. Animals were maintained

on various levels of clofibrate

in the

diet for 2 weeks. Values are mean ? SEM of six rats. Means without common

Statistical Analvsis

Lover Weight Animal

i%)

superscript

are significantly

rats repleted with testosterone in castrated rats (Table 1).

a

(p < .05) different.

was significantly

higher than

Total Peroxisomal P-Oxidation Activity Total peroxisomal P-oxidation activity is used to assess the degree of peroxisomal proliferation.s,” To further delineate the role of testosterone in peroxisomal proliferation, we measured total peroxisomal P-oxidation activity in the liver. Figure 1 shows that clofibrate treatment induced the proliferation of peroxisomes in a dose-dependent manner in all three groups of rats. However, the induction of peroxisomal P-oxidation activity in castrated rats was significantly blunted compared with that in intact rats (Fig 1). When castrated male rats were repleted with testosterone, proliferation of peroxisomes by clofibrate was restored to the level observed in intact male rats (Fig 1). However, this effect of testosterone was noted only at the two lower levels of clofibrate (0.25% and 0.50%). When castrated male rats were administered the highest level of cfofibrate (l.O%), proliferation of peroxisomes was similar in the presence or absence of testosterone (Fig 1). Serum Clofibrate Levels The above results indicated that testosterone increased the sensitivity of peroxisome proliferation by clofibrate, but that the maximal response to clofibrate was unchanged. One explanation for these findings is an effect of testosterone deficiency to enhance clofibrate clearance. To investigate this possibility, we measured serum levels of clofibrate produced by oral feeding in the presence or absence of testosterone. Figure 2 shows that serum clofibrate levels increased progressively in all three groups of rats as dietary levels of clofibrate were increased. However, serum clofibrate levels were significantly lower in castrated male rats than in intact male rats at all three levels of dietary clofibrate (Fig 2). Moreover, when castrated male rats were repleted with testosterone, serum clofibrate levels were restored to levels observed in intact male rats (Fig 2). Serum cfofibrate levels were undetectable in untreated animals.

PAUL, SEKAS, AND WINTERS

170

INTACT

RATS

m

CASTRATED

RATS

r\‘1

CASTRATED

+

TESTOSTERONE

80

de

;

cde

T

80

cd

40

20

0 0

0.25

0.50 %

CLOFIBRATE

Fig 1. Total peroxisomal p-oxidation activity in the liver. Peroxisomal p-oxidation activity was measured in the 600 x CJsupernatant fraction of liver homogenate using the palmitoyCCoA-dependent, potassium cyanide-insensitive, NAD reduction. Each bar represents the mean f SEM of six tats. Means that do not share a common fetter are significantly (P < .05) different

UDP-G’lucllro~~ylt~ansferase Activity

The major pathway for the metabolism of clofibrate in rodents is by glucuronidation.‘l Glucuronidation occurs in the liver and is catalyzed by UDP-glucuronyltransferase. To investigate whether lower serum clofibrate levels observed in castrated male rats were due to increased glucuronidation, the activity of UDP-glucuronyltransferase was measured in liver homogenates using bilirubin as the aglycone. Without clofibrate treatment. there were no significant differences in hepatic UDP-glucuronyltransferase activity between the three groups of rats (Table 2). Treatment of rats with 0.25% clofibrate caused a fourfold to fivefold increase in hepatic UDP-glucuronyltransferase activity (Table 2). Treatment of rats with 0.5% and 1.0% clofibrate caused a further increase in UDP-glucuronyltransfcrase activity (Table 2). However, within each level of clofibrate, there were no significant differences in UDP-glucuronyltransferase activity among the three groups of rats (Table 2). We also measured hepatic UDP-glucuronyltransferase activity using a second substrate, 4-methylumbelliferone. Transferase activity was measured with 4-methylumbclliferone only in rats receiving 0.50% dietary clofibrate. There were no significant differences in UDP-glucuronyltransferase activity with this substrate in livers of three groups of rats (8.63 2 0.45, 7.40 t 0.52, and 7.49 + 0.86 nmol/ minimg protein in intact, castrated, and castrated rats replaced with testosterone, respectively. mean + SEM of six rats). Because of the unavailability of radiolabeled

clofibrate. transfcrasc

we were not able to measure UDP-glucuronylactivity using clofibrate as the substrate. DISCUSSION

The objective of this study was to investigate the role of testosterone in the clofibrate-induced proliferation of peroxisomcs in the liver. The rationale for conducting this investigation follows from the sexual dimorphism that occurs in the responsiveness of rat liver to a number of peroxisome proliferators, including clotibrate:‘“J’ malt animals show greater response than female animals.ZTJ1 Additionally, the incidence of liver tumors, including hepatoccllular carcinoma, is higher in male than in female rats administered clofibrate.” Our study of castrated rats and castrated rats replcted with testosterone suggests that testosterone is the testicular hormone responsible for this sexual dimorphism. The peroxisomal proliferative response was asscsscd by two criteria, namely the changes in total peroxisomal P-oxidation activity and the degree of hepatomegaly. The results of this study show that testosterone plays an important role in the proliferation of hepatic peroxisomcs and in the development of hepatomegaly. This conclusion is supported by three sets of observations. First, total peroxisomal P-oxidation activity, a marker of peroxisomal proliferation in the liver, was significantly decreased in castrated male rats compared with intact male rats. Second. hcpatomcgaly. which often parallels pcroxisomal proliferation, was also

TESTOSTERONE

AND PEROXISOME PROLIFERATION

171

60 INTACT RATS

50

m

CASTRATED

RATS

I\

CASTRATED

+ TESTOSTERONE

40

& $

3o

b T

20

IO

0 0.50

0.25 %

1.00

CLOFIBRATE

Fig 2. Serum clofibrate levels. Serum clofibrate levels were determined spectrophotometrically as described in the Methods. represents the mean + SEM of six rats. Means that do not share a common letter are significantly (P < .05) different.

significantly blunted in castrated male rats compared with intact male rats. Third, when castrated male rats were repleted with testosterone, both the proliferation of peroxisomes and hepatomegaly increased to levels observed in intact male rats. The effect of testosterone could be either to modify the action of clofibrate on peroxisome proliferation or to Table 2. Effect of Clofibrate Treatment UDP-glucuronyltransferase Dietary

UDP-Glucuronyltransferase

Levels

Activity

of Clofibrate 1%)

0

0.26

0.50

1.OO

on

Activity in the Liver

Animal

(nmol/m~nlmg

Group

protein)

Intact male rats

0.068 2 0.018

Castrated male rats

0.067 -c 0.01’

Castrated + testosterone

0.058 2 O.Ola 0.33 r 0.03bC

Intact male rats Castrated male rats

0.25 -t 0.03b

Castrated + testosterone

0.31 -c 0.02bC

Intact male rats

0.41 + 0.03c

Castrated male rats

0.33 2 O.OZbC

Castrated + testosterone

0.38 2 0.04bC

Intact male rats

0.61 f 0.03d

Castrated male rats

0.60 i 0.06d

Castrated + testosterone

0.67 I 0.04d

NOTE. Animals were maintained on various levels of clofibrate for 2 weeks. UDP-glucuronyltransferase

activity was measured in liver ho-

mogenate using bilirubin as the aglycone. Values are means + SEM of six rats. Means without a common superscript are significantly (P < .05) different.

Each bar

influence clofibrate metabolism. We found that serum clofibrate levels were significantly lower in castrated male rats than in intact and testosterone-repleted castrated rats. Consequently. hepatic levels of clofibrate in castrated male rats may not be adequate to induce as large a peroxisomal proliferative response as in intact male rats. The finding that other fibrates arc also more potent peroxisomal proliferators in male than in female rat@ suggests that the metabolism of other drugs in this class may also be affected similarly. By contrast, the development of hepatomegaly following treatment with the androgen precursor steroid dehydroepiandrosterone was similar in male and female mice.‘9 This observation further suggests the role of testosterone in the development of hepatomegaly and peroxisomal proliferation. The fact that the proliferation of peroxisomes and the development of hepatomegaly were blunted in castrated rats at the two lower levels (0.25% and 0.50%) but not at the highest level (1.0%) of clofibrate can be explained as follows: although the metabolism of clofibrate in castrated male rats receiving the highest dietary level of clofibrate is accelerated by androgen deficiency, the level of clofibrate produced in the serum is sufficient to induce the proliferation of peroxisomes. In fact, serum levels of clofibrate in castrated male rats given 1.0% dietary clofibrate were as high as those of intact male rats given 0.50% clofibrate. The major pathway for the metabolism of clofibrate is by glucuronidation in the liver.24 To explain the observed

PAUL, SEKAS, AND WINTERS

172

influence of testosterone on serum clofibrate levels, we measured the activity of UDP-glucuronyltransferase in liver homogenates. No significant differences in the activity of this enzyme were found among the three experimental groups. This was true when either 4-methylumbelliferone or bilirubin were used as substrates to assay this enzyme. These results suggest that lower serum clofibrate levels in castrated male rats may be due to mechanism(s) other than glucuronidation, such as the induction of alternate metabolic pathways or accelerated renal excretion. On the other hand, because multiple UDP-glucuronyltransferases are present in the liver, our results obtained with alternate substrates may not be applicable to the metabolism of clofibrate. Further studies regarding the relationship between gonadal steroids and clofibrate metabolism are warranted. In addition, our results do not exclude the possible decreased intestinal absorption of clofibrate under conditions of androgen deficiency, although such an effect seems unlikely. Specific cytoplasmic hepatic peroxisome proliferatorbinding proteins have been described.3” Additionally, recent reports have identified a peroxisome proliferatoractivated receptor as a member of a novel steroid hormone

receptor superfamily.3’m23 The expression ot pcroxisomc proliferator-activated receptor has been shown to bc tissuespecific, with the highest level of expression in the liver.“’ Liver is also the tissue that is most responsive to peroxisome proliferator$ and has the highest incidence of tumors.” Whether peroxisome proliferator-binding protein or peroxisome proliferator-activated receptor are intluenccd by testosterone is not known. In summary, the results of the present study show that the absence or presence of testosterone may he an important determinant of the serum level of clotibratc produced by oral feeding. We propose that testostcronc’s effects on serum levels of clofibrate account for the different peroxisomal proliferative response of intact and castrated male animals, and by implication, malt versus female rats. Further, the higher incidence of liver tumors in male than in female rats administered clofibrate may be accounted for in part by sex differences in the metabolism of this drug.

ACKNOWLEDGMENT The technical assistance of Lori Pauley and Dushan greatfully acknowledged.

Ghoclray

is

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