the effect of glutathione depletion by buthionine sulphoximine on 1-cyano-3,4-epithiobutane toxicity

Fd Chem. Toxic. Vol. 29, No. 3, pp. 153-157, 1991 0278-6915/91 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1991 Pergamon Press plc

THE EFFECT OF GLUTATHIONE DEPLETION BY BUTHIONINE SULPHOXIMINE ON

1-CYANO-3,4-EPITHIOBUTANE TOXICITY

J. L. VANSTEENHOUSE*, M. J. FETTMAN and D. H. GOULD Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State

University, Fort Collins, CO 80523, USA

(Received 26 June 1990; revisions received 27 November 1990)

Abstract--The effect of glutathione (GSH) depletion by buthionine sulphoximine (BSO) on the nephrotoxicity and GSH-enhancing effect of the naturally occurring, crucifer-derived nitrile 1-cyano-3,4- epithiobutane (CEB), was investigated. Male Fischer 344 rats were administered 50 or 125 mg CEB/kg body weight by gavage with or without prior ip treatment with 550 mg/kg body weight L-BSO. One group of control animals was treated with water only by gavage, while another group was pretreated with BSO and then given water by gavage. Liver and kidney samples were taken 48 hr after CEB treatment for GSH determinations and histological examination. The high-dose CEB without BSO resulted in increased GSH in liver and kidney, marked karyomegaly in the pars recta of renal proximal tubules and tubular epithelial necrosis, which was limited to a few renal tubules. The low-dose CEB alone resulted in increased hepatic GSH and mild karyomegaly. Pretreatment with BSO abrogated the tubular necrosis and karyomegaly induced by either CEB dose. BSO pretreatment inhibited low-dose CEB-induced GSH enhancement in the liver. The combined BSO and high-dose CEB treatment still resulted in increased hepatic GSH, although the increase was less than that observed with high-dose CEB alone. In the kidney, BSO pretreatment abrogated the high-dose CEB-induced increase in GSH, but GSH content was not significantly different from that with high- or low-dose CEB alone. These results provide evidence that CEB conjugation may be a bioactivation reaction with the conjugate involved in nephrotoxicity. The conjugate may also be involved in increasing renal and hepatic GSH.

INTRODUCTION

1-Cyano-3,4-epithiobutane (CEB) is a naturally occurring nitrile derived from glucosinolates present within cruciferous plants (Kirk and MacDonald, 1974). Glucosinolates within the plants decompose under certain conditions of processing or storage to release a variety of thiocyanates, isothiocyanates and nitriles (Kondo et al., 1985; Tookey et al., 1980). Crucifer diets rich in nitriles have been associated with toxic effects in poultry, swine and rats (Bowland et al., 1963; Umemura et al., 1977; VanEtten et al., 1969). Two glucosinolate-derived nitriles, CEB and the closely related 1-cyano-2-hydroxy-3,4-epithiobutane have been isolated and shown to be nephrotoxic in male rats resulting in degeneration and necrosis of proximal tubules located at the tips of medullary rays, renal tubular karyomegaly and impaired renal function (Gould et al., 1985; VanSteenhouse et al., 1989; Wallig et al., 1988). The metabolism of these nitriles has not been studied extensively, but, in addition to being nephrotoxic, increased hepatic glutathione (GSH) and non-protein thiols were observed

*To whom all correspondence should be addressed at: Department of Veterinary Pathology, School of Veteri- nary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.

Abbreviations: BSO = buthionine sulphoximine; CEB = 1- cyano-3,4-epithiobutane; GSH = glutathione; MeCCNU = l - (2-chloroethy l ) -3- ( t rans-4-methy lcyc lohexyl ) - l -

nitrosourea.

in male rats 48 hr after the first of two daily doses (Wallig and Gould, 1986; Wallig et al., 1988). Admin- istration of CEB induces rapid depletion of hepatic GSH, which precedes increased hepatic GSH content (VanSteenhouse et al., 1989). In addition, elevated renal GSH content was evident as early as 4 hr after CEB administration, with no depletion phase observed.

GSH is a ubiquitous tripeptide involved in a number of cellular functions, including the detoxication of potentially toxic, mutagenic and carcinogenic electrophilic xenobiotics. Conjugation with GSH has been proposed as a significant metabolic pathway for CEB as a result of the electrophilic nature of the epithiol moiety, and the observation of rapid hepatic GSH depletion and increased urinary non-protein thiol excretion (VanSteenhouse et al., 1989). The toxicities of compounds such as vinylidene chloride (Andersen and Jenkins, 1977; Jaeger et al., 1974) and acetaminophen (Mitchell et al., 1973) are potentiated by GSH depletion. However, there are instances whereby GSH conjugation results in bioactivation and the formation of metabolites more reactive than the parent compound (Igwe, 1986; Reichert and Schutz, 1986; van Bladeren et aL, 1980).

Nephrotoxicity has been associated with the transport and accumulation of GSH conjugates and their metabolites in the kidney (Elfarra and Anders, 1984). The GSH conjugate, or resultant cysteine conjugate, may be directly toxic or may be cleaved by lyases in the kidney to produce toxic, reactive thiols (Tateishi and Shimizu, 1980). The present study was

153

154 J.L. VANSTEENHOUSE et al.

undertaken to determine the effect, if any, of altered GSH metabolism on the renal GSH content and morphological changes observed in CEB toxicity. Renal and hepatic GSH was depleted in male rats by treatment with buthionine sulphoximine (Griffith, 1981) prior to CEB administration. It was hypothesized that subsequent changes in the pattern of toxicity would aid in determining the roles of the parent compound and the GSH conjugate in CEB toxicity.

MATERIALS AND METHODS

Animal management. Thirty-six male Fischer 344 CDF rats weighing 173.2-200.9 g (189.1 +__ 7.0) were obtained from Charles River Laboratories (Wilming- ton, MA, USA). One wk prior to CEB treatment the rats were fed a semi-purified diet, AIN-76A, with cornstarch substituted for sucrose to prevent hepatic lipidosis (Hamm et al., 1982). Throughout the experimental periods the rats were housed individually in metabolic cages under the controlled conditions of a 12-hr light/dark cycle at 21°C and 30-50% relative humidity. Water was allowed ad lib., but pair-feeding was instituted to account for effects of nutrient intake on hepatic GSH levels. Dietary intake of both the low-dose animals and the controls was restricted to approximate the intake of the animals in the high- dose group, so that comparisons could be made between all three groups. Eighteen rats were pretreated with an ip injection of 550 mg L-buthionine sulphoximine (BSO)/kg body weight in 5 ml/kg body weight sterile phosphate buffered saline, pH 7.35, 2 hr prior to CEB treatment, to reduce the GSH content to 50% and < 10% of normal levels in the liver and kidney, respectively. Three groups (6 rats/group) were given, by gavage, one of the following: 0.5 ml distilled, deionized water; 50rag CEB/kg body weight; or 125 mg CEB/kg body weight. The lower dose of CEB induces mild renal epithelial cell karyomegaly, whereas the higher dose results in renal tubular necrosis and degeneration as well as marked karyomegaly (VanSteenhouse et al., 1989). Another 18 rats were not pretreated with BSO, but were similarly administered, by gavage, either water or, 50 or 125 mg CEB/kg body weight. Forty-eight hr after CEB administration, when CEB-induced lesions are consistently observed, the rats were anaesthetized by ether inhalation, exsanguinated and autopsied. Liver and kidney samples were prepared for histological examination and GSH assays. Urine volumes were recorded and samples were saved frozen at -20°C.

Chemicals. CEB was synthesized according to the protocol of Luthy and Benn (1980). The final product was purified by Kugelrohr distillation. The purity was found to be greater than 98% by using nuclear magnetic resonance. The compound was stored in sealed ampoules in pure form at -15°C. Reduced GSH, glutathione reductase (NADPH: oxidized- glutathione oxidoreductase; EC 1.6.4.2), reduced fl-NADPH and 5,5'-dithiobis-(2-nitrobenzoic acid) were obtained from Sigma Chemical Co. Ltd (St Louis, MO, USA). BSO was obtained from Chemical Dynamics Corp., (South Plainfield, N J, USA) and dietary components from ICN Nutritional Biochemical (Cleveland, OH, USA).

Tissue preparation. Tissue samples taken at autopsy for histological examination were fixed in 10% neutral buffered formalin. Fixed tissues were trimmed, dehydrated in graded ethanol, embedded in paraffin, cut at 4/~m and stained with haematoxylin and eosin. Liver and kidney tissue samples (approxi- mately 150-200 mg each) were placed in 15-ml glass tubes containing 4 ml 0.02 M-EDTA for GSH determinations. These samples were then homogenized for 30see using a Tissumizer * rotor-stator generator (Tekmar, Cincinnati, OH, USA) and 4ml 10% trichloroacetic acid was added. Homogenized, de- proteinized samples were kept on ice until centrifuged for 15 rain at 3000 g. The supernatants were removed and then bubbled with nitrogen to remove oxygen and prevent oxidation of GSH, then sealed and stored at -20°C until analysed.

Biochemical assays. GSH determinations were performed on the supernatants using the colorimetric recycling method of Tietze (1969). All colorimetric enzyme activity assays were performed on a Gilford 250 spectrophotometer (Gilford Instruments, Ober- lin, OH, USA) equipped with a flow-through cell (Rapid Sampler # 2443-A).

Histological examination. Haematoxylin and eosin- stained tissue sections were examined for lesions and used for renal karyometry. Areas containing straight portions of proximal tubules at tips of medullary rays were outlined with ink using a stereomicroscope. One-hundred and twenty nuclei of proximal tubular epithelial cells within these fields, excluding affected cells with pyknotic nuclei, were measured on an Olympus BH-2 microscope, magnification × 1000, using the semi-automated Bioquant Hypad Digitizer system and data program (R&M Biometrics, Nashville, TN, USA).

Statistical analysis. GSH values were evaluated by analysis of variance with respect to treatment group, and the means of each group were related to one another using Fisher's least significant difference test for multiple comparisons at a 95% confidence level (Snedecor and Cochran, 1967). Mean renal proximal tubule epithelial nuclear size and frequency distributions of nuclear size were determined for each animal, with subsequent mean nuclear size and frequency distributions for respective treatment groups calculated. The means of the number of nuclei in respective size ranges were then compared with one another, relative to treatment, by analysis of variance using Fisher's least significant difference test. Nuclear size means were also compared by analysis of variance relative to treatment group.

RESULTS

Liver GSH

Animals treated with either dose of CEB alone exhibited increased (P < 0.05) hepatic GSH. The increase with the high dose was significantly greater (P < 0.05) than with the low dose. Pretreatment with BSO inhibited the GSH-enhancing effect of the low- dose CEB. BSO followed by the high-dose CEB did exhibit increased (P < 0.05) hepatic GSH, but the increase was significantly less (P < 0.05) than with high-dose CEB alone (Fig. 1).

Glutathione depletion and CEB toxicity 155

Renal GSH

Only those animals treated with high-dose CEB alone had increased (P < 0.05) renal GSH. Treat- ment with BSO alone resulted in GSH values significantly less (P <0.05) than controls. BSO pretreatment followed by low-dose CEB resulted in GSH values significantly less (P < 0.05) than with low-dose CEB alone. The results of the combined BSO and high-dose CEB treatment were more complex. Pretreatment with BSO did inhibit the enhanced GSH induced by the high-dose CEB alone. GSH in the BSO-pretreated high-dose group was not significantly increased relative to non-BSO-pretreated control, and it was not significantly different from that for either high- or low-dose CEB alone, but was greater (P < 0.05) than for other BSO-pretreated groups (Fig. 2).

Histological examination

Rare angular, eosinophilic pars recta renal tubular epithelial cells with small, condensed nuclei were observed in 5 of the 6 rats treated with high-dose CEB alone. Pars recta renal tubular epithelial karyomegaly was also detected in all animals in the same group. No microscopic renal lesions were observed in the low-dose CEB group or any BSO pretreated groups.

In this study, as in previous studies (VanSteen- house et al., 1989; Wallig et al., 1988), there were no hepatic histological lesions.

Karyometry for pars recta renal tubular epithelial cells

Animals treated with low-dose CEB alone demonstrated an increased (P < 0.05) mean nuclear size (40.98/am2). The distribution of nuclei was shifted towards larger nuclei, with fewer (P < 0.05) nuclei measured in the 25-35/~m 2 range and greater num- bers (P < 0.05) found between 35 and 65 #m 2. Ani- mals treated with the high-dose CEB alone exhibited a mean nuclear size (55.90/~m 2) greater (P < 0.05) than both control (33.80/~m:) and low-dose CEB groups. In the high-dose CEB alone group the num-

"• 8

o 4 E

T 2

lo ~t

o ControLs 50mg/kg CEB 125 mg/k9 CEB

T r e a t m e n t .

Fig. 1. Effect of CEB on rat liver GSH levels after 48 hr, with and without prior GSH depletion by BSO. Values shown are means + SEM (n = 6 per group). • = P < 0.05 v. controls; t =greater than respective BSO-pretreated group, P < 0.05; *= P < 0.05 v. BSO-pretreated control

and low-dose CEB groups.

4,

2

E

T ~ 4

• + Bso [ ] - BSO

ControLs

0

t

50 rag/k9 CEB 125mg/kg CEB

Tr eo t men t

Fig. 2. Effect of CEB on rat kidney GSH levels after 48 hr, with and without prior GSH depletion by BSO. Values shown are means + SEM (n = 6 per group). • = P < 0.05 v. controls; t =greater than respective BSO-pretreated group, P < 0.05; *= P < 0.05 v. BSO-pretreated control

and low-dose CEB groups.

ber of nuclei measured in the smaller size ranges (<45/zm 2) was significantly less (P < 0.05) and an increased (P < 0.05) number of nuclei was measured in the range greater than 45/~m 2 (Fig. 3). Pretreat- merit with BSO inhibited both the increase in mean nuclear size and the shift in frequency distribution towards larger nuclei induced by either dose of CEB alone. Values for all BSO-pretreated groups were comparable with controls (Fig. 3).

DISCUSSION

A single 125 mg/kg body weight dose of CEB resulted in karyomegaly and necrosis of scattered individual tubular epithelial cells in the pars recta of proximal tubules located at the apex of medullary rays. Marked karyomegaly, quantitatively evident as an increased mean nuclear size and distinct variations in size frequency distribution, was demonstrated in renal proximal tubules in animals treated with the high-dose CEB. Mild karyomegaly was also observed in animals treated with the lower 50 mg/kg body weight dose, which in this, and in previous studies (VanSteenhouse et al., 1989), induced no demonstra- ble necrosis.

BSO depletes GSH in tissues by inhibiting the rate-limiting enzyme for GSH synthesis, ~,-glutamyl- cysteine synthetase (Griffith, 1981). Pretreatment with BSO abrogated the toxic effects of the high-dose CEB, as illustrated by the lack of histological or karyometric changes in pretreated animals. These results are in contrast to the enhanced nephrotoxicity of 1-(2-chloroethyl)-3-( trans-4-methyicyclohexyl)- l- nitrosourea (MeCCNU) after depletion of GSH by BSO (Kramer et al., 1985). The protective effect of GSH against MeCCNU was further demonstrated by the novel appearance of focal areas of centrilobular hepatic necrosis after depletion. GSH depletion affording protection against toxicity, as seen with

156 J.L. VANS~NHOUSE et al.

8 0 -

~) 4- BSO +/cont.roL / \ -~- BSG+/Low CEB / \ -,- Bso+/h iQ. CEB

60" ' ~ 3 ' \ ~I- BSO-/Low CEB '~ )l~ll \ T -0- BSO-/high CEB

c 4 0

E

~' 2O

t . . . . . ; - - ~ , ~ . :;3 . ~

d d d e NucLear a r e a (/J.m z)

Fig. 3. Frequency distribution of nuclear size of pars recta proximal tubule cells in rats treated with CEB, with and without prior GSH depletion by BSO. Data points shown are means _+ SEM (n = 6 rats per group, 120 nuclei/rat). ~), = Greater than control and BSO pretreated groups, P < 0.05; 1" =greater than all other treatment groups,

P < 0.05.

CEB, suggests GSH conjugation may result in bioactivation rather than detoxication. The absence of CEB-induced liver lesions in GSH-depleted rats also suggests that the parent compound is not directly toxic in its unaltered state.

Glutathione conjugates have been implicated in the nephrotoxicity of several halogenated hydrocarbons. The nephrotoxicity of GSH conjugates of 1,2-dibromoethane and 1,2-dichloroethane have been attributed to the formation of a reactive episulphonium ion intermediate (Elfarra et al., 1985; Guengerich et al., 1987; Webb et aL, 1987). Similarly, the nephrotoxicities of hexachlorobutadiene and tri- chloroethylene have been associated with GSH conjugation, but by a different mechanism. With these compounds, there is evidence for cleavage of the resultant cysteine conjugate by renal fl-lyase, resulting in the formation of a reactive thiol (Elfarra et al., 1986; Nash et al., 1984).

Interpretation of the effects of BSO pretreatment on the GSH-enhancing effect of CEB is complex. In this study, in animals treated with BSO alone, the return of hepatic GSH to levels comparable with controls illustrates the transient effect of BSO, and the liver's remarkable capability for synthesis and replenishment of GSH. Hepatic GSH in the rats pretreated with BSO, followed by either dose of CEB, was less than that in the rats treated with CEB alone. Increased hepatic GSH induced by low-dose CEB was inhibited by BSO pretreatment. BSO-pretreated rats administered the high-dose CEB demonstrated less hepatic GSH than with high-dose CEB alone, but this was increased relative to controls and to other BSO-pretreated groups. Various studies demonstrat- ing a ' rebound' elevation in GSH following depletion have consistently shown the extent of elevation to be relative to the extent of depletion (Cote et al., 1984; D'Souza et al., 1988; Nakagawa et al., 1984). A

similar pattern of dose-dependent hepatic GSH depletion followed by increased GSH was demonstrated with CEB (VanSteenhouse et aL, 1989). Depletion of GSH would be expected to be greatest in those rats treated with both BSO and high-dose CEB, yet CEB alone resulted in greater GSH elevation. At this time, 48 hr after BSO administration, the inhibitory effect of BSO would be expected to have diminished, but may contribute to lower GSH levels in animals receiving the combined treatment. However, the significant elevation in hepatic GSH levels in BSO- pretreated animals when combined with the higher CEB dose may suggest a CEB-induced mechanism for increasing GSH synthesis independent of the extent of prior GSH depletion.

In our study, in animals treated only with BSO, renal GSH was significantly lower than control values, which signifies more extensive depletion of renal GSH and/or a lower rate of GSH synthesis in the kidney than in the liver. As in the liver, animals pretreated with BSO followed by high-dose CEB exhibited increased renal GSH compared with other BSO pretreated groups. However, this was not significantly different from GSH values obtained for either the high or low doses of CEB alone. This further suggests an undetermined CEB-induced mechanism for stimulating GSH synthesis. As discussed above, BSO pretreatment masked CEB-induced karyomegaly at both doses, which was attributed to decreasing the delivery of GSH conjugate to the kidney. Extensive GSH depletion by BSO could also conceivably inhibit development of renal karyomegaly through a direct effect on DNA synthesis, rather than as a result of decreased formation and transport of GSH conjugate. Glutathione is involved in information of the active site of ribonucleotide reductase, which catalyses the reduction of ribo- nucleotides to deoxyribonucleotides (Stryer, 1988). Glutathione also functions as a coenzyme in the reduction of methylglyoxal to lactate, and it has been hypothesized that accumulation of methylglyoxal in the absence of GSH could retard cell growth (Meister, 1988). Thus, depletion of GSH could result in a decreased ability to increase DNA synthesis, even in the presence of a toxicant. However, in this study, karyomegaly was inhibited by BSO pretreatment prior to high-dose CEB. This occurred despite renal GSH values that were comparable with those in both the high- and low-dose CEB groups, which did exhibit karyomegaly.

We have thus presented evidence that conjugation of CEB with GSH may be an intermediary bioactivation step, rather than one of detoxication, with the GSH conjugate involved in CEB-induced nephrotoxicity. The identity and nature of the conjugate, or its subsequent metabolite, remain to be determined. We also suggest that the conjugate, directly or indirectly, is involved in increasing hepatic and renal GSH through an undetermined mechanism.

Acknowledgements--The authors wish to thank Joe Savage and Dr Frank Stermitz of the Colorado State University Chemistry Department for synthesizing and providing the CEB. This work was funded by NIEHS 5T32ES07152, USDA-SEA W-122 and Colorado Experiment Station.

Glutathione depletion and CEB toxicity 157

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the effect of glutathione depletion by buthionine sulphoximine on 1-cyano-3,4-epithiobutane toxicity

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