Changes in enzyme activities in the urine and tissues of rats fed sodium o-phenylphenate

Fd Chem. Toxic. Vol. 22, no. 5, pp. 361-364, 1984

0278-6915/84 $3.00+0.00 Copyright © 1984 Pergamon Press Ltd

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CHANGES IN ENZYME ACTIVITIES IN THE URINE A N D TISSUES OF RATS FED SODIUM o-PHENYLPHENATE F. NAGAI and T. NAKAO Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Hyakunin-cho 3-24-1, Shinjuku-ku, Tokyo 160, Japan (Received 7 June 1983)

Abstract--The activities of certain enzymes in the urine and tissues of rats given 2% sodium ophenylphenate in the diet for 20 wk were examined. Urinary ~,-glutamyl transpeptidase (~GTP) decreased immediately after the start of feeding of the treated diet and its activity remained low for 20 wk. The 7GTP and alkaline phosphatase (ALP) activities in kidney homogenate decreased to about 80% of the control at 20 wk, but G6PD activity was significantly increased; Na,K-ATPase was unchanged. On the other hand, the ~GTP activity in the liver homogenate of treated rats was increased to about eight times that of the controls, the G6PD activity showed a significant increase, but the ALP and Na, K-ATPase activities were not significantly different from the control values. The glutathione concentration in the livers of treated rats was significantly reduced.

INTRODUCTION

Sodium o-phenylphenate (OPP-Na) has been approved in Japan for use as a fungicide on products for human consumption, and has been used as such on some imported citrus fruits. Hiraga & Fujii (1981) reported that tumours of the urinary system were induced by long-term administration of diets containing OPP-Na to rats (Hiraga & Fujii, 1981). However, OPP-Na had no teratogenic or mutagenic effects in mice (Ogata, Ando, Kubo & Hiraga, 1978; Ogata, Yoshida, Nawai et al. 1978), and no chromosomal aberrations attributable to OPP-Na were detected in CHO-KI cells (Nawai, Yoshida, Nakao & Hiraga, 1979; Yoshida, Nawai & Hiraga, 1979). Nakao et al. recently reported studies of the metabolic profile of OPP-Na in rats (Nakao, Ushiyama, Kabashima et al. 1983). 7GTP is a loosely membrane-bound glycoprotein, and is widely distributed in mammalian tissues. The enzyme is said to catalyse the transfer of the ~-glutamyl group from glutathione and several other substrates to various amino acid and peptide acceptors (Meister & Tate, 1976). In adult mammalian tissues, the activity of this enzyme is highest in the kidney, followed by the pancreas, while the level in the liver is very low. However, ~,GTP activity is increased in hepatoma and preneoplastic liver nodules, and foetal liver shows high activity (Albert, Orlowska, Orlowski & Szewczuk, 1964; Albert, Rzucidlo & Starzyk, 1970; Cameron, Kellen, Kolin et al. 1978; Fiala & Fiala, 1973; Fiala, Fiala & Dixon,

Abbreviations: ALP

=Alkaline phosphatase; G6PD= glucose-6-phosphate dehydrogenase; ~GTP = y-glutamyl transpeptidase; OPP-Na = sodium o-phenylphenate; PNPP = p-nitrophenylphosphate disodium salt. 361

1972; Fiala, Mohindru, Kettering, et al. 1976; Taniguchi, Tsukada, Mukuo & Hirai, 1974). Urinary 7GTP activity is relatively high, and it has been suggested that this is a result of the high level of activity in the kidney (Rosalki, 1975). ALP is also a membrane-bound enzyme, and has frequently been assayed to aid clinical diagnosis. Increased G6PD activity in the liver has been reported to be induced by the administration of various hepatotoxins (Kojima, Hama & Kubodera, 1981; McLeamn, Reid & Gurney, 1964; Watanabe & Taketa, 1973). Changes in the glutathione content of foetal liver, hepatoma and hepatotoxin-treated liver have also been reported (Fiala et al. 1976; Taniguchi et al. 1974). This paper describes changes in urinary 7GTP activity and in the activities of certain enzymes in the liver and kidneys of rats fed 2% OPP-Na for 20 wk. EXPERIMENTAL

M a t e r i a l s . OPP-Na was technical grade Dowicide A (Lot No. NM 010445, 95% pure) obtained from Dow Chemical Co., Midland, MI, USA. The 7GTP assay kit (Code 271-32702) was obtained from Wako Pure Chemical Industries Ltd, Osaka. p Nitrophenylphosphate disodium salt was purchased from Tokyo Kasei Co. Glucose-6-phosphate monosodium salt (No. 7879) was obtained from Sigma Chemical Co., St Louis, MO, USA. A n i m a l s . Male and female 4-wk-old F344 DuCrj (Fisher) rats were obtained from Charles River Japan Inc., Kanagawa. The animal room was maintained at 25 _+ I°C, with a relative humidity of 5 5 _ 5%. Fluorescent lighting was controlled to give a 12-hr light-dark cycle. Diet. OPP-Na was mixed with powdered diet CE12 (Nihon Clea Co. Ltd, Tokyo), at a concentration of 2%, and the mixture was made into pellets. After

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'F. NAGAI and T. NAKAO

prefeeding with the control diet for one week, the rats were divided into two groups, each comprising five males a n d five females. The rats were fed control diet or diet c o n t a i n i n g 2~o O P P - N a for 136 days. Diet a n d water were supplied ad lib. Enzyme assays. 7 G T P activity was m e a s u r e d with L-y-glutamyl-~-naphthylamide as substrate using a n assay kit. One unit o f enzyme activity was defined as the a m o u n t t h a t released 1/~mol ct-naphthylamide per m i n u t e at 37°C. The effects of O P P - N a concentrations from 1 x 10-SM to 2 x 10-2M o n urinary YG T P activity in vitro were also measured. Alkaline p h o s p h a t a s e was assayed as follows. The enzyme was i n c u b a t e d in 500/~1 o f assay mixture c o n t a i n i n g 20 m M - p - n i t r o p h e n y l p h o s p h a t e disodium salt (PNPP), 5 mM-MgCI a n d 50 mM glycine-HC1 (pH 10) at 37°C. T h e reaction was t e r m i n a t e d by a d d i n g 1 ml of 0.1 N-NaOH, a n d the a m o u n t of p - n i t r o p h e n o l liberated from P N P P was determined color±metrically at 410 n m with a Gilford 300N s p e c t r o p h o t o m e t e r . Na, K - A T P a s e was assayed according to the m e t h o d o f N a k a o , N a k a o , Mizuno, et al. 1973). Glucose-6p h o s p h a t e dehydrogenase ( G 6 P D ) was m e a s u r e d spectrophotometrically at 37°C using a Cary 17 spectrometer (Kojima et al. 1981). Glutathione determination. The c o n c e n t r a t i o n o f g l u t a t h i o n e in tissues was determined by the m e t h o d of Owens & Belcher (1965). Sample preparation. R a t urine was collected in glass metabolism cages for 6 h r per day a n d kept in a n ice b a t h as described previously ( N a k a o et al., 1983). T h e volume was m e a s u r e d a n d the urine was dialysed in Visking t u b i n g overnight at 4°C against distilled water in three changes. The dialysed urine was stored at - 8 0 ° C until required. After nearly 20 wk of feeding, the control a n d experimental groups were killed by decapitation. The liver, bladder a n d a kidney were r e m o v e d immediately, a n d stored at - 8 0 ° C until required. The liver was h o m o g e n i z e d in a Teflon h o m o g e n i z e r in five

volumes of ice-cold 250 mM-sucrose a n d 4 mM-TrisE D T A (pH 7.4). Kidney a n d bladder homogenization was p e r f o r m e d similarly except t h a t a P o l y t r o n h o m o g e n i z e r ( K i n e m a t i c a G m b H , Lucerne, Switzerland) was used. Liver h o m o g e n a t e was centrifuged at 2400 g in a Sorvall RC-2B centrifuge using a SS-34 r o t o r at 4°C; the pellet (P1) was suspended in the h o m o g e n i z i n g medium. The s u p e r n a t a n t was centrifuged at 105,000g for 6 0 m i n in a B e c k m a n 40A rotor; the pellet (P2) was suspended in the h o m o g e nizing medium. T h e s u p e r n a t a n t (S) was also retained. T h e protein c o n c e n t r a t i o n s were determined according to the m e t h o d o f Lowry, R o s e b r o u g h , F a r r & R a n d a l l (1951).

RESULTS U r i n a r y ) , G T P activity gradually increased during the period o f feeding o n control diet, a n d in controls was higher in males t h a n in f e m a l e s (Table 1). However, urinary ),GTP activities decreased immediately after the start o f feeding o f the diet containing O P P - N a in b o t h sexes, a n d m u c h reduced activities c o m p a r e d with the controls were m a i n t a i n e d t h r o u g h o u t the feeding study. The activities of y G T P a n d A L P in kidney h o m o genate were reduced to a b o u t 80~o o f the control levels after 20 wk o n the test diet (Table 2). G 6 P D was considerably elevated, but Na, K - A T P a s e was unchanged. G l u t h a t i o n e was not detectable in the kidney by this method. T h e liver weight was increased in treated rats c o m p a r e d with the controls (Table 3). Table 3 also shows t h a t y G T P activity (per gram of liver) was increased to a b o u t eight times that in control livers a n d G 6 P D activity was significantly increased. The c o n c e n t r a t i o n of g l u t a t h i o n e w a s reduced in the liver of rats fed O P P - N a , b u t A L P a n d Na, K - A T P a s e activities in the liver were unchanged.

Table 1. Effect of feeding 2~o OPP-Na in the diet for 20wk on rat urinary ?GTP activity Activity of yGTP in urine of Control group Duration of feeding

nmol/ml/min*

0days 1 day 3 days 1 wk 2 wk 5 wk 10 wk 18 wk

70.7 _ 7.7 58.1 -+-4.8 79.8 ± 4.8 213.8 ± 19.3 389.9 ± 64.3 474.6 ± 64.3 394.8 _ 112.9 531.8 ± 140.4

Total't Males 70.6 + 6.0 61.1 + 13.8 63.2 ± 22.9 259.5 ± 59.2 484.3 __.151.4 587.6 + 61.0 608.6 _ 203.8 662.8 ± 279.2

2~o OPP-Na group nmol/ml/min*

Total'~

76.9 + 15.3 41.5 + 14.6 16.5 ± 4.2 43.1 ± 9.4 76.9 ± 16.2 93.6 ± 41.8 90.9 ± 15.6 84.3 ± 19.6

57.9 _ 21.1 48.8 + 17.2 24.0 ± 9.3 70.8 _ 33.2 143.6 + 28.1 90.3 + 51.1 201.5 + 54.0 117.2 ± 45.8

Females

0 days 33.0 _ 8.3 21.6 ± 9.2 36.4 ± 2.5 33.4 ± 9.5 1 day 52.6 ± 8.1 48.9 ± 22.6 29.0 ± 3.0 37.9 ± 4.4 3 days 58.9 + 12.0 55.6 ± 6.9 14.6 ± 5.8 24.0 ± 11.4 1 wk 76.5 ± 14.6 96.4 ± 12.8 17.3 ± 1.2 32.4 ± 2.7 2 wk 148.4 ± 26.8 186.8 ± 50.4 24.1 ± 4.8 40.6 + 10.6 5 wk 71.4 ± 37.4 132.2 ± 24.1 7.0 ± 4.2 14.8 ± 6.7 10 wk 89.6 ± 146.6 146.5 ± 81.6 19.5 ± 4.9 36.2 ± 9.2 18 wk 177.1 ± 96.2 166.1 ± 71.2 8.7 ± 4.7 11.7 __.8.9 Values are means ___SD for groups of five rats. *nmol ct-naphthylamide/ml urine/min. tThe total activity value is the ),GTP activity nmol ~t-naphthylamide/minof urine collected for 6 hr.

363

Effects o f O P P - N a o n e n z y m e activities Table 2. Effect of feeding 2~o OPP-Na in the diet for 20 wk on the weight, enzyme activities and glutathione content of rat kidneys Kidney parameter

Sex

Control group

Weight (g) Weight (g) 1'GTP activityt 1'GTP activityt ALP activity3~ ALP activity:~ G6PD activity§ G6PD activity§ Na,K-ATPase activityll Na,K-ATPase activityll Glutathionee concn Glutathione concn

M F M F M F M F M F M F

2.86 ± 0.166(5) 1.285 + 0.034(5) 100.4 ± 3.7(5) 87.0 + 3.7(5) 1093.2 + 108.7(5) 1861.3 ± 160.5(5) 0.485 + 0.049(5) 0.270 + 0.033(4) 1688.7 ± 165.7(5) 1639.9 + 44.0(5) ND ND

2~o OPP-Na group 2.348 ± 0.145(5) 1.277 ± 0.0068(5) 81.9 ± 3.4(5)*** 67.7 + 3.9(5)*** 951.6 ± 88.5(5)* 1506.9 + 126.8(7)** 0.759 + 0.049(4)*** 0.327 ± 0.045(4)* 1488.0 ± 368.2(5) 1526.4 ± 189.3(5) ND ND

ND = Not detected t#mol ~-naphthylamide/g kidney/min. :~#mol PNP/g kidney/hr. §#mol substrate transformed/g kidney/min. II#mol inorganic phosphate/g kidney/hr. Values are means ± SD for the number of assays indicated in parentheses; those marked with asterisks differ significantly (Student's t test) from the corresponding control value *P < 0.1; **P < 0.01; ***P < 0.001). All assays were performed on homogenates.

ALP activity in the rat bladder was also assayed but was not significantly affected by administration of OPP-Na (data not shown). DISCUSSION

Rat urinary ),GTP activity decreased within a few hours of the start of OPP-Na feeding, and the reduced activity was maintained during 20 wk of feeding. The concentration of OPP-Na in the dialyzed urine used in this assay was less than 1 x 10-3 M, and urinary 7GTP activity was not reduced by 2 x 10-3M-OPP-Na in vitro. Urinary ALP was previously shown to be decreased in rats fed OPP-Na (Kobayashi, Kabashima & Nakao, 1982), and in the present study yGTP and ALP in the kidneys of treated rats were decreased to about 80~o of the control levels. Rat renal y GTP is localized on the lumenal surface of the brush border membrane of renal tubules (Meister & Tate, 1976). Urinary yGTP is considered to be released from the lumenal surface during turn-over of renal epithelial cells (Rosalki,

1975). ALP is also bound to the brush border membrane. Histological examination showed that pyelonephritis occurred in rats fed subchronic amounts of OPP-Na (Hiraga & Fuji±, 1981). Our biochemical data suggest that administration of OPP-Na damages the renal tubules. In the livers of rats fed 2~o OPP-Na for 20 wk, we observed a considerable elevation of ), GTP activity, a significant increase of G6PD activity and a concomitant decrease of glutathione content. Ushiyama et al. have previously reported the induction of cytochrome P-450 in the livers of rats fed OPP-Na for 1 wk (Ushiyama, Fujitani, Nagai et al. 1982). 7GTP activity is low in healthy mammalian adult liver, but high in foetal liver, hepatoma, preneoplastic liver nodules and in hepatitis victims (Albert et al. 1964; Albert et al. 1970; Cameron et al. 1978; Fiala et al. 1972; Fiala et al. 1973; Fiala et al. 1976; Taniguchi et al. 1974). The yGTP activity of rat hepatomas induced by azo dyes have been investigated in detail by Taniguchi et al. (Taniguchi, 1974; Taniguchi, Saito & Takakuwa, 1975). Further,

Table 3. Effect of feeding 2 ~ OPP-Na in the diet for 20wk on the weight, enzyme activities and glutathione content of rat liver Liver parameter

Sex

Control group

Weight (g) Weight (g) "yGTP activityt ~,GTP activityt ALP activity~ ALP activity:~ G6PD activity§ G6PD activity§ Na,K-ATPase activityll Na,K-ATPase activityll Glutathione concn¶ Glutathione concn¶

M F M F M F M F M F M F

10.31 ± 0.51(5) 5.97 _+ 0.51(5) 46.1 ± 13.9(5) 63.8 ± 33.5(5) 66.5 ± 10.6(5) 46.5 ± 10.6(5) 1.063 _ 0.076(5) 2.691 ± 0.184(5) 1.44 ± 0.44(5) 1.91 + 0.53(5) 7.24 ± 0.85(5) 4.84 ± 1.07(5)

2~o OPP-Na group 12.06 + 0.63(5)** 6.19 __.0.26(5)** 357.4 ± 20.4(5)* ** 495.9 _ 9.9(5)*** 56.1 ± 13.5(5) 49.6 ± 10.4(5) 1.628 ± 0.131(4)*** 2.920 ± 0.076(4)* 1.69 ± 0.35(3) 1.68 ± 0.53(4) 3.60 ± 0.53(4)*** 1.89 ± 0.53(5)**

tnmol ct-naphthylamide/g liver/min. :~/~mol PNP/g liver/hr. §#mol substrate transformed/g liver/min. II,umol inorganic phosphate/rag protein/hr. ¶#mol/g liver. Values are means + SD for the number of assays indicated in parentheses; those marked with asterisks differ significantly (Student's t test) from the corresponding control value (*P <0.1; **P < 0.01; ***P < 0.001). Fraction 'P2' was used for assays of Na,K-ATPase, and 'S' was used for the G6PD assay. The rest of the assays were performed on the homogenate.

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increases in the liver glutathione levels of rats given various chemical carcinogens or tumour cells have been reported (Fiala et al. 1976; Taniguchi et al. 1974). This elevation of 7 G T P activity may be significant in the process of chemical carcinogenesis in rat liver. However no hepatomas have yet been observed histologically in rats fed 2% O P P - N a (T. Fujii, personal communication, 1981). The problem requires further study. We have also investigated the effect of the concentration of O P P - N a in the diet. Rat urinary y G T P decreased dose-dependently with increasing concentrations of O P P - N a in the diet, but the striking increase of liver 7 G T P activity was observed only for 2% O P P - N a in the diet; 0.25%, 0.5% and 1%, OPPN a did not have this effect (Nagai & Nakao, 1983).

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