TOXICOLOGYANDAPPLIEDPHARMACOLOGY42, 571--581 (1977)

The Effects of Nitrofuran Derivatives on Hepatic Microsomal Mixed-Function Oxidase Activity in Rats I MORIO FUKUHARA2 AND EIGO TAKABATAKE The Institute of Public Health, Shirokanedai, Tokyo, 108, Japan Received March 16, 1977; accepted May 18, 1977

The Effects of Nitrofuran Derivatives on Hepatic Microsomal Mixed-Function Oxidase Activity in Rats. Ft:KUHARA,M., ANDTAKABATAKE,E. (1977). Toxicol. AppL Pharmacol. 42, 571-581. The effects of four nitrofuran antibacterial food additives and animal drugs were studied on the mixed-functionoxidase activityin rat hepatic microsomes. Dietary administration (0.03 to 0.3%) of the compounds for 7 days to Wistar male rats, produced varying degrees of liver enlargement and some compounds exhibited a reducing effect on cytochrome P-450 concentrations and NADPH-cytochrome c reductase activity in hepatic microsomes. The activity of aminopyrineN-demethylase and anilinehydroxylase was affected in varying ways by these compounds. Among these, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide(furylfuramide)was most potent in increasing the liver weight and in reducing the activity of these microsomal enzymes. In male rats fed a 0.1% furylfuramide-containing diet, the cytochrome P-450 concentration and the activity of aminopyrine N-demethylase and aniline hydroxylase were markedly reduced throughout the experimentalperiod up to 29 days, while cytochrome b5 and the activity of NADPH-cytochrome c reductase were not changed so markedly. Similar results were also obtained in female rats fed the furylfuramide diet for 3 days, but the reduction in enzyme activitieswas smaller than that in male rats. Dietary administration of furylfuramidefor 3 days to phenobarbital- or 3-methylcholanthrene-pretreatedfemale rats resulted in a marked decrease in the activity of the microsomal enzymes. The pentobarbital sleeping time was prolonged by 57% in male rats fed a 0.1% furylfuramidediet for 7 days. The incubation in vitro of furylfuramide (0.05 mM) with microsomes from livers of nontreated male rats resulted in decreased activity of aminopyrineN-demethylaseand anilinehydroxylase. Nitrofuran derivatives have been widely used as antibacterial drugs in chickens, turkeys, and swine to fight disease and promote growth. These include nitrofurazone, furazolidone, nihydrazone, furaltadone, panazon, and furamizole, which have been recently banned as feed additives for animals in J a p a n and the United States. Furylfuramide, a nitrofuran derivative which had been exclusively used in Japan since 1965 as a preservative for foods such as soybean curd and processed fish products, was also banned in 1974 by the Ministry of Health and Welfare of Japan because of its possible carcinogenic action in laboratory animals. Several preceding investigations had reported that furylfuramide has a mutagenic effect in microorganisms (Kada, 1973; K o n d o and Ichikawa-Ryo, 1973) and a potential to induce chromosomal aberration in cultured h u m a n lymphocytes and bone marrow cells ( T o n o m u r a and Sasaki, 1973). 1A preliminary report of this work was published in Chem. Pharm. Bull (Tokyo) 23, 1626-1628 (1975). 2Send reprint requests to Dr. Morio Fukuhara, The Institute of Public Health, 6-1, Shirokanedai 4 chome, Minato-ku, Tokyo, 108, Japan. Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

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However, apart from the carcinogenicity, few studies have disclosed other toxic effects of furylfuramide to laboratory animals. Though the possible action on testis atrophy has been discussed (Miyaji, 1971a), no teratogenic effect has been observed with furylfuramide (Miyaji, 1971b; Imahori, 1975). On the other hand, a significant liver hypertrophy has been generally observed in the animals treated with furylfuramide (Aiso et al., 1964; Miyaji, 1971a), but its significance has not been well defined. Many chemicals that produce liver enlargement are known to induce the activity of the microsomal mixed-function oxidase system, which suggests the adaptive response of liver to xenobiotics (Golberg, 1966). Miyaji (1971a) also deduced that the hypertrophy of liver cells induced by furylfuramide might be the consequence of the enhanced function to metabolize the ingested compound. On the other hand, few compounds are known to depress the activity of the microsomal mixed-function oxidase system for any duration of time without demonstrating any pathological lesions in the enlarged liver. In this report, we present the evidence that furylfuramide and some other nitrofuran derivatives produce a marked liver enlargement without inducing the activity of the microsomal mixed-function oxidase system. METHODS

Chemicals) Nitrofuran derivatives used were 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide(furylfuramide), 2-amio-5-[ 1-(2-furyl)-2- (5-nitro-2-furyl)vinyl]-1,3,4-oxadiazole(furamizole), 5-nitro-2-furfurylidene-3-amino-2-oxazolidone(furazolidone), and bis(5nitro,furfurylidene)acetoneguanylhydrazone hydrochloride(panazon). Animals and treatment ofnitrofurans. Animals employed were 5-week-old SPF rats of Wistar-SLC strain, 4 except in the 29-day treatment in which 3-week-old rats were used. Nitrofuran derivatives were mixed into a standard laboratory powdered diet 5 which was given ad libitum. Control rats were fed the standard powdered diet without the respective nitrofuran derivative. Body weight gain and food consumption was recorded during the experimental period. The doses used in the comparative study of these derivatives on microsomal mixed-function oxidase activity were near the maximal tolerated dose in rats which would not significantly affect body weight gain in the treated animals. The dietary levels of the compounds and the period of treatment in each experiment are indicated in the Results section. Pretreatment with phenobarbital and 3-methylcholanthrene. Female rats received ip injections of phenobarbital (80 mg/kg, once daily) or of 3-methylcholanthrene (40 • mg/kg, once daily) for 8 consecutive days and were fed a 0.1% furylfuramide diet for the last 3 days. Pentobarbital sleeping time test. Male rats fed a 0.1% furylfuramide diet for 7 days received ip injection of sodium pentobarbital at a dose of 60 mg/kg and the sleeping time was determined. In vitro incubation with furylfuramide. In vitro effects of furylfuramide on the activity of aminopyrine N-demethylase and aniline hydroxylase were studied using the 3Furylfuramide (Ueno Pharmaceutical Co, Ltd., Osaka) was kindly donated by Dr. Y. Izumo (The Institute of Public Health) and Dr. T. Shiratori (Tokyo Kasei College). Furamizole, furazolidone,and panazon were generous donations of Ueno Pharmaceutical Co. Ltd., Osaka, and Toyama Kagaku Co. Ltd., Tokyo. 4ShizuokaLaboratoriesCo., Hamamatsu. 5Type CE-2, Nippon Clear Inc., Ltd., Tokyo.

NITROFURANS ON MIXED-FUNCTION OXIDASES

573

same assay method as in the in vivo study except that furylfuramide was added to the incUbation media. Microsomes were prepared from livers of nontreated male rats and the enzyme activity of this microsomal preparation was measured in the presence or absence of furylfuramide which was added to the incubation media as a 1.5 mM solution in ethanol-water (1 : 1, v/v) at a final concentration of 0.05 raM. The incubation was carried out for 15 min at 37°C. Assay 6 of mixed-function oxidase activity. The animals were sacrificed at the end of each experiment and the livers were removed immediately, washed with cold saline, and homogenized with 4 vol of 0.25 M sucrose solution (pH 7.4) in a Teflon-glass homogenizer. The hepatic microsomes were isolated from the homogenates by the CaZ+-bound method of Kamath and Rubin (1972). The obtained microsomal precipitates were resuspended in a 0.15 M KC1-0.05 M Tris-HC1 buffer solution (pH 7.4). The microsomal protein was determined by a modification of Lowry's method (Miller, 1961) with bovine serum albumin as a standard. The content of cytochrome P450 and b 5 was measured by the method of Omura and Sato (1964). The activities of NADPH-cytochrome c reductase, aminopyrine N-demethylase, and aniline hydroxylase were assayed by the methods described by Mazel (1972). The contents of cytochromes are expressed as nanomoles per milligram of microsomal protein. The activities of the enzymes are expressed as nanomoles of reduced cytochrome c, nanomoles of formaldehyde released from aminopyrine, and nanomoles of paminophenol formed from aniline per minute per milligram of microsomal protein for NADPH-cytochrome e reductase, aminopyrine N-demethylase, and aniline hydroxylase, respectively. The means and SE of the means were calculated and the levels of significance between the treated groups and the appropriate controls were determined using Student's t-test. RESULTS

Dose-response relationship on the relative liver weight (RLW) and microsomal eytoehrome P-450 concentration. The dose-response relationship of the nitrofurans on RLW (wet weight of liver per 100 g body weight) and on cytochrome P-450 concentrations in hepatic microsomes was determined in male rats treated for 7 days with the derivatives at dietary levels ranging from 0.03 to 0.3%. As shown in Fig. 1, furylfuramide was most potent in increasing RLW among the compounds tested. Furamizole and furazolidone were less potent than furylfuramide, and panazon did not show any effects on RLW. Furylfuramide was also most effective in reducing the cytochrome P-450 concentrations (Fig. 2) and furamizole was effective only at a dietary level of 0.3%, while panazon and furazolidone did not show any effects on cytochrome P-450. Furazolidone increased cytochrome P-450 at a dietary level of 0.1% but it retarded the body weight gain of the treated animals at this dose. Nitrofurans on mixed-function oxidase activity. The effects of nitrofuran derivatives on microsomal mixed-function oxidase activity were studied in male rats fed the diet containing nitrofuran derivatives for 7 days (Table 1). Furamizole slightly decreased, 6Glucose-6-phosphate,NADP, glucose-6-phosphatedehydrogenase,and cytochromee were purchased from Boehringer-Mannheim-YamanouchiCo. Ltd., Tokyo. Aminopyrineand anilinewere obtained from Sanko SeiyakuKogyo Co. Ltd., Tokyo and Ueno ChemicalIndustries,Osaka, respectively.

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though without significant differences, the content of cytochrome P-450 at a dietary level of 0.1%, while the content of cytochrome b 5 was increased significantly. The activity of NADPHicytochrome c reductase was markedly decreased, but that of aminopyrine N-demethylase and aniline hydroxylase was elevated. Furazolidone (0.06% diet) did n o t affect cytochrome P-450 concentrations, but it increased cytochrome b 5. The activities of NADPH-cytochrome c reductase and aminopyrine Ndemethylase were decreased, but that of aniline hydroxylase was increased, by furazolidone. Panazon (0.1% diet) slightly reduced the content of cytochrome P-450. The activities of the other three enzymes were decreased by panazon. Furylfuramide on microsomal mixed-function oxidase activity. The results of furylfuramide treatment on microsomal mixed-function oxidase activity as a function of time are shown in Table 2. The furylfuramide administration at a dietary level of 0.1% to male rats did not retard the body weight gain or reduce the food intake throughout ~o o

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