Toxicology Letters, 55 (1991) 249-254 @ 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0378-4274191~$3.50 ~~O~ISO378427491~~2Q

TOXLET 02510

249

Induction of hepatic peroxisomes by a new, non-carboxylate-containing drug, bifonazole

Shuichi Horiel*, Nobutaka FukumorP and Tetsuya Sugal

‘Department of Clinical Biochemistry, Tokyo College of Pharmacy and =Department of Toxicology. Tokyo

Metropolitan Research Laboratory of Public Health, Tokyo (Japan)

(Received 10 March 1990) (Accepted 15 September 1990)

Key words: Peroxisomes; /?-Oxidation; Hypolipidemic drug; Bifonazoie; Receptor

SUMMARY

The acute effect of an antimycotic drug, bifonazole, on hepatic peroxisomes of rats was studied in com- parison with that of clotrimazob, which has a similar structure. By feeding 0.5% bifonazole in the diet for 5 days, the activities of camitine acyltransferase, carnitine ~lmitoyltransferase and the ~roxisomal &oxidation system were increased by 30-, 3- and 7-fold, respectively, over the control. Under the same conditions, clotrimazole did not cause such changes. Electron microscopic observation showed that perox- isome proliferation had been induced by bifonazole treatment. Thus, a compound which does not contain a carboxylate moiety can induce peroxisomes in rodent liver.

INTRODUCTION

I-~,~-Diphenylbenzyl)imidazole (bifonazole, Bay h 4502, Mycospori@, Bayer, Le- verkusen) is a new antifungal imidazole derivative [l-3] with the structural formula shown in Figure 1. Its antifungal activity is more potent than those of the first imida- zoyl antimycotics to be discovered, clotrimazole and miconazole [2].

Several hypolipidemic drugs and certain phthalate-ester plasticizers, when fed to rodents and certain non-rodent species, cause an increase in the activity of peroxiso- ma1 ~-oxidation and proliferation of peroxisomes in the liver [4-S]. All of these per-

*Present address and correspondence: Shuichi Horie, Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan.

Abbreviations: bifonazole = I-@,a-diphenylbenzyl)imidazole; clotrimazole = l-(o-chloro-a,a-diphenyl- benzyl)imidazole; CAT = camitine acetyltransferase; CPT = camitine palmitoyltransferase; POS = per- oxisomal b-oxidation system; TG = triacylglycerol.

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(A) (3 (8) Fig. 1. Structural formulae in bifonazole (A) and clotrimazole (B).

oxisome-proliferating agents consist of a carboxylic function carried on an hydro-

phobic backbone, although the carboxylic function may be either present initially in

the agents or derived by metabolic oxidation of the respective alcohol or aldehyde

[9]. Hertz et al. [9] defined the requirement of an amphipathic carboxylate in initiating

peroxisomal proliferation.

The relationship between the structures of peroxisome proliferators and that of pu-

tative receptor protein, as well as the mechanism of hypolipidemic action of these

agents, is discussed in this paper.

MATERIALS AND METHODS

Bifonazole and clotrimazole were donated by Bayer Research Centre, Wuppertal

(F.R.G.). Male Wistar rats weighing 18&21Og were fed ad libitum with a powdered

laboratory diet containing 0.5 s (w/w) bifonazole or clotrimazole for 5 or 10 days.

Liver homogenates were prepared and the activities of catalase, carnitine acetyltrans-

ferase (CAT), carnitine palmitoyltransferase (CPT) and the peroxisomal P-oxidation

system (POS) were measured as described previously [lo]. Protein content was deter-

mined by the method of Lowry et al. [l 11. Triacylglycerol (TG) content in the liver

and serum was determined [lo]. Samples for electron-microscopic observation were prepared as described pre-

viously [12] and morphological analysis was performed by scanning randomly se-

lected fields in a Hitachi HU- 12A electron microscope [ 121.

RESULTS

Table I shows the effects of bifonazole and clotrimazole on the hepatic enzyme ac-

tivities and some other parameters. In the rats fed on bifonazole diets, marked in-

creases in the activities of POS and CAT were observed. The activity of CPT was

also increased by the same treatment whereas catalase activity did not change signifi-

cantly. Dose-dependent changes in these enzyme activities were recognized. When

we measured the time course of the changes in the activities of POS, CAT and CPT

after the feeding of 0.2% bifonazole to rats, the activities were found to increase with

the feeding in d time-dependent manner (data not shown). The administration of clo-

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TABLE I

EFFECTS OF BIFONAZOLE AND CLOTRIMAZOLE ON HEPATIC ENZYME ACTIVITIES AND OTHER PARAMETERS IN THE LIVER AND SERUM OF RATS

Control Bifonazole

0.2% 0.5%

Clotrimazole

0.2% 0.514

Catalase 49.1 i5.7 (unit/g liver) (1)

FAOS 483k 109 (unit/g liver) (1)

CAT 252+89 (unit/g liver) (1)

CPT lOlO* (unit/g liver) (1)

Liver weight (% of body wt.)

Liver TG

(mgig) Serum TG

(mg/dl) Protein

(mg/g liver)

4.55i0.21

(1) 4.1+ 1.0

(1) 56.4k20.4

(1) 19717

(1)

59.4511.2 (1.21)* 2289+206 (4.74)** 4818+391 (19.12)** 1909+341 (1.89)**

5.6lkO.29 (1.23)** 4.5*0.5 (l.lo)* 29.2k8.1 (0.52)* 215k 19 (I.o9)*

70.8 f 14.6 (1.44)** 3412k I75 (7.06)** 7869k492 (31.23)‘* 3095&257 (3.06)**

7.49kO.35 (1.65)** 5.9kO.3 (1.44)” 11.3+4.1 (0.20)** 254+27 (1.29)+*

38.314.9 (0.78); 440+ 121 (0.91) 471+136 (1.87) 990+121 (0.98)

5.02&0.31 (l.lo)* 11.9k4.7 (2.90)** 27.Ok6.3 (0.48)** 211,l (1.07)**

18.li5.6 (0.37)** 348+115 (0.72)* 817+162 (3.24)** 1065k 118 (1.05)

6.03iO.40 (1.33)** 18.4+ 11.6 (4.04)* 12.1 i4.6 (0.21)** 217& 10 (l.Io)**

Rats were fed a diet containing 0.2 or 0.5% (w/w) bifonazole or clotrimazole for 5 days. Control group received standard diet. Results are expressed as means f SD of 4 rats. Relative values compared with the control are given in parentheses. Statistical evaluations were performed by using Student’s t-test. *P<O.O5, **P<O.Ol, vs. control.

trimazole increased the CAT activity, although the increase in the ratio to the control was only approximately one-tenth of that of the bifonazole-treated rats. POS and CAT activities were not significantly changed by the treatment and catalase activity was decreased to 37 % of the control. On the other hand, hepatomegaly was observed in rats treated with both agents. Significant decreases in serum TG were found in rats given diets containing both agents, but serious hepatic lipidosis was seen in the case of clotrimazole-treated rats. The administration of 0.2% bifonazoie in the diet to rats caused a 50% decrease in serum TG content without any significant reduction of body weight compared with the control.

Figure 2 shows electron micrographs of liver sections. Marked proliferation of he- patic peroxisomes was observed in the rats treated with bifonazole. Peroxisomes without a nucleoid core tended to be more abundant than in the control rats. The induction of liver peroxisomes was dependent on the amount of bifonazole fed to the rat.

Fig. 2. The induction of hepatic peroxisomes by bifonazole in vivo. Male rats were treated with bifonazole. (a) control; (b) 0.248 (w/w) bifonazole for 10 days; (c) 0.5% (w/w) bifonazole for 10 days. The liver semi- sections were stained with many1 acetate and lead citrate. In (b) and (c), most peroxisomes contain a nucleoid core, but anucleoid peroxisomes are also seen frequently. Note the appearance of large perox- isomes. (d) 0.2% (w/w) bifonazole for 5 days. Peroxisomes were stained by 3,3-diaminobenzidine [26].

P = peroxisomes. x 6440 (a,b,c); x 2760 (d).

DISCUSSION

Proliferation of peroxisomes caused by the administration of bifonazole is similar in nature to that induced by other peroxisome proliferators such as clofibrate [13], nafenopin 1141, tibric acid [5], WY-14,643 [S], di-(2-ethylhexyl)phthalate [15] and others [7]. All of these are hydrophobic compounds which contain a carboxylate moiety in the structure. Bar-Tana et al. [9,16] showed that ~,~-methyl-substituted dicarboxylic acids exhibit potent h~olipidemic action and induction of hepatic per- oxisome proliferation. They suggested that the free carboxylic function or a deriva- tive was directly involved in the inductive process, while the nature of the hydropho- bic backbone might determine the efficacy [9]. However, bifonazole, which is a peroxisome inducer, does not contain carboxylate and there is no plausible route for its metabolism to an amphipatic carboxylate. It should be noted that the cellular concentration of the agent in different tissues may be an importan factor for the in-

253

duction of peroxisomes. When we determined the distribution of radioactivity in rat tissues after oral administration of [i4C]bifonazole, the concentration of radioactivity was relatively high in the liver compared with other tissues, although the elimination was rapid (unpublished data).

The treatment with bifonazole also increased the activity of CPT 3-fold. This result does not support the proposal by Eacho and Foxworthy [17] that the inhibition of mitochondrial fatty acid oxidation may be involved in the induction of peroxisome proliferation. It is possible that the increase in peroxisomal p-oxidation is an adapta- tion process to a perturbation in lipid metabolism. In fact, the increase in peroxiso- ma1 /?-oxidation activity induced by administration of bifonazole was accompanied by a decrease in the serum TG level, as occurs with other potent peroxisome-inducing agents [ 18-201. The administration of hypolipidemic agents to rodents is predictably associated with hepatomegaly, the extent of which is dependent upon the agent in- volved [4. IO,2 11, and bifonazole is not an exception.

The coordinate induction of peroxisomal /?-oxidation enzymes by structurally dif- ferent compounds implies the existence of a common control mechanism [22]. The peroxisome proliferation may be initiated by the interaction of a peroxisome prolif- erator with a specific cellular binding protein [8,23,24], although Milton et al. [25] rejected the presence of such a binding protein in liver homogenate. In the present study, hepatic peroxisomes were found to be induced by bifonazole but not by clo- trimazole, suggesting that the specific hydrophobic part of the compound is impor- tant for the interaction with the putative binding protein.

The present results show that the carboxylate moiety is not essential for the induc- tion of hepatic peroxisomes and enzymes of the peroxisomal p-oxidation system.

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