Fd Chem. Toxic. Vol. 30, No. 3, pp. 247 250, 1992 0278-6915/92 $5.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1992 Pergamon Press Ltd

SEX D I F F E R E N C E IN THE NEPHROTOXICITY OF T H I A B E N D A Z O L E IN MICE DEPLETED OF

G L U T A T H I O N E BY T R E A T M E N T WITH DL-BUTHIONINE S U L P H O X I M I N E

T. MIZUTANI, K. YOSHIDA, K. ITO and S. KAWAZOE Department of Food Science and Nutrition, Kyoto Prefectural University, Kyoto 606, Japan

(Accepted 26 November 1991)

Alrstract--ln ICR mice depleted of glutathione (GSH) by treatment with DL-buthionine sulphoximine (BSO), males were much more susceptible to thiabendazole (TBZ) nephrotoxicity than females. The nephrotoxicity was indicated by increases in relative kidney weight and serum urea nitrogen (SUN) concentration and by a decrease in renal GSH concentration at 24 hr after TBZ administration. The susceptibility of males to TBZ-induced nephrotoxicity was completely eliminated by pretreatment with oestradiol (OD). Castration of male mice also reduced, though not completely, their susceptibility to TBZ nephrotoxicity. In females pretreated with testosterone (TS), the nephrotoxic effect of TBZ was increased to an extent comparable with that in males.

INTRODUCTION

Thiabendazole (TBZ) is an anthelmintic used in the treatment of various parasite infections in domestic animals and humans. Moreover, TBZ has an anti- fungal activity and has been used as a food preserva- tive and an agricultural fungicide. We have reported that TBZ causes severe kidney injury in male mice depleted of glutathione (GSH) by pretreatment with DL-buthionine sulphoximine (BSO), an inhibitor of GSH synthesis (Mizutani et al., 1990).

In mice, sex differences in renal toxicity and carcinogenicity have been demonstrated for several chemicals such as chloroform (Eschenbrenner and Miller, 1945) and N-nitrosodimethylamine (Noronha, 1977). In the present study, the nephrotox- icity of TBZ was investigated in male and female mice depleted of GSH.

MATERIALS AND METHODS

Chemicals. TBZ was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan) and recrystallized from ethanol. Testosterone (TS) propionate and oestradiol (OD) benzoate were purchased from Wako Pure Chemical Industries (Osaka, Japan). BSO was syn- thesized according to the method of Griffith and Meister (1979).

Animals. Male and female ICR mice, 7 wk old, were obtained from Charles River Japan, Inc. (Kana- gawa, Japan) and used after 1 wk of acclimatization. Mice were housed in aluminium boxes on wood-chip

Abbreviations: BSO = DL-buthionine sulphoximine; OD = oestradiol; GSH = glutathione; SUN = serum urea nitrogen; TBZ = thiabendazole; TS = testosterone.

bedding (White Flake, Charles River Japan, Inc.) at a constant temperature (23 + 2°C) and under a 12-hr light/dark cycle. Except when stated otherwise, mice received food (Funabashi F-2; Funabashi Farms, Chiba, Japan) and water ad lib.

8-wk-old male mice were castrated under pento- barbital anaesthesia and allowed to recover for 18 days before TBZ administration.

Assessment o f nephrotoxicity. Mice were treated ip (between 8 and 9 hr) with BSO (4 mmol/kg body weight) in water at 0.2ml/10g body weight, l hr later, the treated animals were given 200 or 400 mg TBZ/kg body weight in olive oil (0.1 ml/10 g body weight) by oral intubation. The animals were fasted for 16 hr before dosing with TBZ and then for 2 hr after TBZ administration. TS (20 mg/kg body weight) or OD (1 mg/kg body weight) in olive oil (0.03 ml/10 g body weight) was injected sc daily for 7 days before TBZ administration. Mice were killed under pentobarbital anaesthesia; blood was collected by cardiac puncture and the kidney was removed and weighed. The blood was allowed to clot at 37°C for 1 hr, and serum was prepared by centrifugation. Serum urea nitrogen (SUN) concentrations were measured with a commercial kit (Urea NB-test; Wako Pure Chemical Industries). For tissue GSH assay, the whole kidney was homogenized in 8 ml ice-cold 20 mM-EDTA. It has been shown that more than 95% of the acid-soluble sulphhydryl content in mouse tissue could be attributed to GSH, as determined by a more specific procedure (Ip, 1984). Therefore, tissue GSH contents were assessed by determining non-protein sulphhydryl contents according to the method of Sedlak and Lindsay (1968).

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248 T. M1ZUTANI el al.

Table 1. Effects of TBZ in combination with BSO on relative kidney weight, SUN, and renal GSH in male and female mice

Relative kidney weight SUN Renal GSH

Treatment* (% of body weight) (mg%) (,umol/g)

Males Saline + olive oil 2.02 +_ 0.02 21.9 + 1.4 2.96 +_ 0.17 BSO + olive oil 1.88 ± 0.06 21.7 + 2.5 2.21 ± 0.23 BSO + TBZ (200mg/kg) 2.37 ± 0.08t$ 244+_ 11.9++ + 0.56 ± 0.03t++ BSO +TBZ (400mg/kg) 2.37 ± 0.06t:~ 241 + 21.6t++ 0.46 ± 0.03t++

Females Saline + olive oil 1.76 + 0.05 25.3 + 1.4 2.35 ± 0.06 BSO + olive oil 1.62 _+ 0.05 18.6 + 1.1 1.54 + 0.07t BSO + TBZ (200 mg/kg) 1.78 ± 0.05 21.6 ± 2.4 2.13 ± 0.06 BSO + TBZ (400 mg/kg) 1.64 ± 0.11 50.6 ± 20.4 2.39 ± 0.23++

*Mice were treated (as appropriate) with BSO (4 mmol/kg body weight, ip) and then TBZ (orally). Values are means ± SEM for four to six mice; those marked with symbols differ significantly from: tthe

saline + olive oil group value (P < 0.05); ++the BSO + olive oil group value (P < 0.05).

Sta t i s t i ca l analysis . The significance of the differ- ences among multiple groups was determined by Scheffe's test. Compar isons between two groups were made by Student 's t-test. Differences were considered significant at P < 0.05.

RESULTS

Table 1 shows the effects of the t reatment of both male and female mice with TBZ in combinat ion with BSO on relative kidney weight, SUN concentrat ion, and renal GSH concentrat ion 24 hr after dosing with TBZ. In male mice, both 200 and 400 mg TBZ/kg body weight in combinat ion with BSO produced significant increases in relative kidney weight and SUN concentra t ion as previously observed (Mizutani et al., 1990). Fur thermore , these changes were ac- companied by a marked decrease in renal GSH concentrat ion. At this time point, t reatment with BSO alone had no significant effect on renal GSH concen- tration. Previously, we have reported that the treat- ment of male mice with TBZ (up to 1200 mg/kg body weight) alone causes no nephrotoxicity, as judged by normal relative kidney weight and SUN concentra t ion levels (Mizutani et al., 1990). In contrast to males, the t reatment of female mice with either dose of TBZ in combinat ion with BSO did not produce any signifi- cant changes in relative kidney weight and SUN concentrat ion, al though there was a tendency for SUN concentra t ion to be slightly increased following t reatment with the higher dose of TBZ (400 mg/kg body weight). Females treated with BSO alone had a slightly but significantly lower renal GSH concen- trat ion than controls. Treatment with 400 but not

200 mg TBZ/kg body weight in combinat ion with BSO resulted in a higher renal GSH concentra t ion as compared with t reatment with BSO alone. On gross examination, the kidneys of males treated with either dose of TBZ in combinat ion with BSO were enlarged and showed a whitish discoloration. The cut surface showed a whitish band in the cortex. In females, the administrat ion of TBZ in combinat ion with BSO had no such effects on kidney morphology.

The effect of castration on the susceptibility of males to the nephrotoxic effect of TBZ in combinat ion with BSO is shown in Table 2. In castrated mice, t reatment with TBZ (400 mg/kg body weight) resulted in a significant increase only in relative kidney weight. Al though there was a tendency for SUN concen- tration to be slightly elevated, SUN and renal GSH concentrat ions were not significantly altered. On gross examination, the kidneys of only two out of six mice were enlarged and discoloured. In contrast , a 400mg/kg body weight dose of TBZ resulted in marked changes in relative kidney weight, SUN concentrat ion, and renal GSH concentrat ion in nor- mal male mice (Table 1). Taken together, these results indicate that castration tends to reduce the suscepti- bility to TBZ nephrotoxicity.

Table 3 shows the effect of TS pretreatment on the susceptibility of female mice to the nephrotoxic effect of TBZ in combinat ion with BSO. Pretreatment

with TS sensitized females to the toxic effect of TBZ (200mg/kg body weight), as indicated by in- creases in relative kidney weight and SUN concen- tration and by a decrease in renal GSH concentrat ion. The kidneys of all mice in this group appeared pale and swollen.

Table 2. Effects of TBZ in combination with BSO on relative kidney weight, SUN, and renal GSH in castrated male mice

Relative kidney weight SUN Renal GSH

Treatment* (% of body weight) (mg%) (~mol/g)

BSO + olive oil 1.47 + 0.06 20.4 ± 1.2 1.36 ± 0.07 BSO + TBZ 1.83 _ 0.lit 79.3 + 38.0 1.19 ± 0.26

*Castrated mice were treated with BSO (4 mmol/kg body weight, ip) and then received either TBZ (400 mg/kg body weight, orally) or olive oil alone.

Values are means __+ SEM for five mice. The value marked with a dagger differs significantly from that for the BSO + olive oil group (P < 0.05).

Sex difference in TBZ nephrotoxicity in mice

Table 3. Effects of TBZ in combination with BSO on relative kidney weight, SUN, and renal GSH in female mice pretreated with testosterone (TS)

Relative kidney weight SUN Renal GSH

Treatment* (% of body weight) (mg%) (/~mol/g)

BSO + olive oil 1.60 ± 0.09 19.1 ± 0.5 3.64 ± 0.15 BSO + TBZ 1.98 ± 0.09¢ 130 ± 27. I t 1.41 ± 0.50t

*Mice were pretreated with TS (20 mg/kg body weight, sc) daily for 7 days. The pretreated mice were treated with BSO (4 mmol/kg body weight, ip) and then received either TBZ (200 mg/kg body weight, orally) or olive oil alone.

Values are means + SEM for five or six mice, those marked with daggers differ significantly from those for the BSO + olive oil group (P < 0.05).

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Table 4. Effects of TBZ in combination with BSO on relative kidney weight, SUN, and renal GSH in male mice pretreated with oestradiol (OD)

Relative kidney weight SUN Renal GSH

Treatment* (% of body weight) (rag%) (#mol/g)

BSO + olive oil 1.78 ± 0.05 19.8 ± 1.0 2.76 ± 0.15 BSO + TBZ 1.67 ± 0.07 19.4 ± 2.2 3.31 ± 0.24

*Mice were pretreated with OD (1 mg/kg body weight, sc) daily for 7 days. The pretreated mice were treated with BSO (4 mmol/kg body weight, ip) and then received either TBZ (400 mg/kg body weight, orally) or olive oil alone.

Values are means + SEM for five or six mice.

Table 4 shows the effect o f O D pre t rea tment on the renal toxicity of T B Z in combina t ion with BSO in male mice. O D completely protected males from the nephro toxic effect of T B Z (400 mg/kg body weight), as judged by the practically normal kidney weight, S U N and renal G S H concent ra t ions , and gross appearance of the kidneys.

DISCUSSION

In a previous study, we have shown that the increases in relative kidney weight and SU N concen- t ra t ion in mice treated with T B Z in combina t ion with BSO parallel the severity of tubu la r necrosis (Mizutani et al., 1990). In addi t ion, the present study shows that the increases in relative kidney weight and S U N concen t ra t ion were always accompanied by a marked renal G S H deplet ion (Table 1). In the present study, changes in relative kidney weight, S U N concen t ra t ion and renal G S H concen t ra t ion were therefore used as indices of nephrotoxici ty .

The present s tudy clearly shows that in ICR mice only males are susceptible to the nephrotoxic i ty caused by TBZ in combina t ion with BSO (Table 1). The susceptibili ty of males to TBZ-induced nephro- toxicity was markedly reduced by cas t ra t ion (Table 2). In contrast , in females t reated with TS the nephro toxic effect of T B Z was increased to an extent comparab le with tha t observed in males (Table 3). These results suggest tha t the sex difference in T B Z nephrotoxic i ty is related to an effect of TS. The lack of TBZ nephrotoxic i ty in males pre t reated with O D (Table 4) may result f rom the an tagoniz ing effect of O D at the level of androgen receptors in the kidney or f rom the depression of androgen p roduc t ion by O D in the testis.

In mice, ch lo roform produces renal injury only in males and not in females (Eschenbrenner and Miller,

1945). A series of experiments in ICR mice has shown that renal cy tochrome P-450 metabolizes ch loroform to a nephrotoxic intermediate, and that the sex difference in nephrotoxic i ty appears to be related to differences in renal cy tochrome P-450 concent ra t ions and mixed-funct ion oxidase activities, which are regulated by androgenic hormones (Smith and Hook, 1984; Smith et al., 1984). Our previous data suggested that the cy tochrome P-450-media ted metabol ism of TBZ to a reactive intermediate was a necessary step in inducing nephrotoxici ty in GSH-deple ted mice (Mizutani et al., 1990). Therefore, the sex difference in TBZ- induced nephrotoxici ty observed in the pre- sent study may be explained, at least in part , by a possible difference in cy tochrome P-450-dependent metabol i sm of TBZ in the kidney between the two sexes.

Recently, Hi rayama et al. (1987) have demon- s t rated that the half-lives of G S H in the liver and kidney are significantly shor ter in male than in female C57BL/6NJcl mice, as determined after inhibi t ion of GSH synthesis by t rea tment with BSO. This observat ion suggests tha t BSO pre t rea tment produces a more extensive deplet ion of GSH in males than in females, and that this difference in G S H level between the two sexes results in the sex-related difference in the nephrotoxic i ty of TBZ in combina t ion with BSO. However, this possibility is ra ther unlikely because at 3 hr after admin is t ra t ion of BSO alone renal G S H concent ra t ions were not significantly different between male and female ICR mice, whereas hepatic G S H was higher in males than in females (data not shown).

Acknowledgements--We would like to thank Ms T. Iguchi, Ms T. lkeda and Ms S. Kinefuchi for their excellent technical assistance.

250 T. MIZUTANI et al,

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