Species differences in the nephrotoxic response to S-(1,2-dichlorovinyl)glutathione

Toxicology Letters, 60 (I 992) 343-35 1 0 1992 Elsevier Science Publishers B.V. All rights reserved 037%4274/92/$05.00

TOXLET

343

027 11

Species differences in the nephrotoxic response to S-( 1,2-dichlorovinyl)glutathione

Saroj Chakrabarti, D@wtement

M. Anwar Malick, Claude Denniel and E. Greselin

de Mtidecine du Truwil et Hygihe

du Milieu, FucultP de Medecine, Universiit! de MonthI.

QuChec (Canudu) (Received

9 October

(Accepted

16 December

1991) 1991)

Key words: S-( 1,2-Dichlorovinyl)glutathione;

Nephrotoxicity;

Species differences;

Renal metabolism

SUMMARY The present

study was carried

S-( 1,2-dichlorovinyl)glutathione

out to investigate (DCVG)

the species differences

using rats, hamsters

in the nephrotoxic

and guinea-pigs.

response

to

DCVG was given intrape-

ritoneally in physiological saline to groups of 5 animals at doses 0, 165 and 330 pmol/kg. Urine was collected for 24 h and the animals were then sacrificed. Significantly increased levels of urinary glucose, N-acetyl-p-p-glucosaminidase, in rats at both

y-glutamyl

dose levels of DCVG.

transpeptidase, An increase,

proteins

and blood urea nitrogen

but not of similar

magnitude,

were observed

of these biochemical

parameters was noted in hamsters only at the higher dose of DCVG. Guinea-pigs showed significant increases in these biochemical parameters at the lower dose, but not at the higher dose. Light-microscopic studies showed increasing proximal tubular necrosis (PTN) in rats with increasing dose of DCVG, but PTN involving straight tubules only was observed at the higher dose in hamsters. PTN was again observed in guinea-pigs

at the lower dose, but not at the higher dose of DCVG.

INTRODUCTION

Glutathione conjugation is an important cellular defence mechanism against a wide variety of reactive electrophiles. The S-glutathione conjugate formed initially is subsequently degraded to the S-cysteine conjugate which in turn is N-acetylated to form the N-acetyl-S-cysteine conjugate, a mercapturic acid [ 11. The majority of conjugates formed by this pathway are non-toxic, but evidence is accumulating that GSH conjugates of a variety of compounds and/or their corresponding cysteine conjugates are

Correspondence

to. Dr. S. Chakrabarti,

cult& de Medecine, 357.

Universite

Departement

de Montreal.

de Medecine

P.O. Box 6128, Station

du Travail

et Hygiene

‘A’, Montreal,

Quebec,

du Milieu, FaCanada

H3C

344

nephrotoxic [2-41. S-( 1,2-Dichlorovinyl)glutathione (DCVG), a glutathione conjugate of trichloroethylene, is nephrotoxic in rats [5], in isolated rat proximal tubular cells [6] and in LLC-PKI cells in culture [7]. The toxicity of DCVG is blocked by inhibitors of y-glutamyl transferase (y-GT) and of cysteinyl glycine dipeptidase and aminopeptidase, showing the importance of its metabolism to the cysteine conjugate for the expression of nephrotoxicity. Moreover, the nephrotoxicity of DCVG is blocked by amino-oxyacetic acid, an inhibitor of P-lyase, indicating the necessity for its further metabolism to a toxic thiol by the pyridoxal phosphate-dependent /?-lyase. Species differences in renal I/-GT activity is highest in rats, intermediate in mice and lowest in guinea-pigs [8]. On the other hand, guinea-pigs form mercapturic acids much less readily than do rats or rabbits [9]. Thus, species differences in the specific activity of the mercapturic acid pathway enzymes may determine the susceptibility of a given GSH-S-conjugate-induced nephrotoxicity. Furthermore, there are significant physiological differences between species in proximal tubular structure and function [lo]. The present study was therefore carried out to investigate the species differences in the nephrotoxic response to DCVG using rats, hamsters and guinea-pigs. MATERIALS

AND METHODS

S-( 1,2-Dichlorovinyl)glutathione (DCVG) was synthesized according to the method of McKinney et al. [I 1] and its purity was greater than 99%. All other chemicals were of reagent quality. Male Fischer-344 rats (180&200 g), hamsters (160-l 80 g) and guinea-pigs (650-670 g) were obtained from Charles River Canada, Inc. (St. Constant, Quebec). They were maintained in a temperature-controlled room with a 12-h light/dark cycle and had continuous access to water and appropriate Laboratory Chow. All animals were placed in metabolism cages 5 days before treatment. DCVG was mixed in physiological saline and injected intraperitoneally to groups of 5 animals at doses 0, 165 and 330 pmolikg. Urine was collected over ice from the animals housed in metabolism cages during O-24 h after the administration of vehicle or DCVG and the animals were then sacrificed for blood withdrawal and kidney removal. Biochemical parameters Urinary glucose was estimated with Sigma kit no. 115, based on the method described by Carroll et al. [12], The urinary activity of y-glutamyl transpeptidase (yGT) was assayed calorimetrically [13]. The urinary activity of N-acetyl-/?-D-glucosaminidase was determined using a calorimetric method [ 141 following filtration of the urine on a Sephadex G-50 column [15]. Urinary proteins were estimated according to the calorimetric method of Bradford [ 161 and blood urea nitrogen using Sigma kit no. 535. Histopathology Tissue sections

of kidneys

of control

and DCVG-treated

animals

were processed

345

routinely, embedded in paraffin, sectioned at 4-6 ,um, stained with hematoxylin eosin and by the periodic acid-Schiff method and examined by light microscopy.

and

Statistics

Comparisons between multiple-treatment groups (each groups of 5 animals) were performed using analysis of variance, completely randomized design, and treatment means were compared using Dunnett’s t-test, with P < 0.05 as the criterion of significance. RESULTS

The results of measurements showing the influence of DCVG on several biochemical parameters characteristic of renal injury in different animal species 24 h after its administration are presented in Figures l-5. The urinary glucose was 10 and 12 times higher at 165 and 330 ,umol/kg body wt. respectively in DCVG-treated rats than that of control rats (Fig. 1). The hamsters showed a 4.9-fold increase in urinary glucose excretion only at the highest dose level, whereas guinea-pigs showed no such significant increase, but a decrease at the highest dose (Fig. 1). There was no increase in plasma glucose concentrations (data not shown here) due to such DCVG treatment on any animal species studied in this work. Urinary excretion of y-GT was increased 7-fold due to DCVG at 165 pmolikg, while at 330 pmollkg such excretion was increased S-fold in rats (Fig. 2). In hamsters, there was no increase in the excretion of

l=l Rats

*

n

T

-

m

it Hamsten,

Control 165 ~ole/kg 1xn ..-rl-/I,”

L

Guinea pigr

Fig. 1. Influence of intraperitoneal treatment with DCVG on the urinary excretion of glucose at 24 h. Results are averages i SE determinations made on separate groups of 5 animals. Asterisks indicate values significantly different from the control.

346

240

LT

Control

m m

165 Fole/kg 330 /unole/kg

160

Hamsters Fig. 2. Influence

Guinea pigs

of intrapcritoneal treatment with DCVG on the urinary excretion of y-glutamyl peptidase (y-CT) at 24 h. For further explanation, see Fig. I.

trans-

y-CT at the lower dose level of DCVG, while a significant increase (4.8-fold) in urinary y-GT was seen at the higher dose level (Fig. 2). On the other hand, urinary excretion of y-GT in guinea-pigs was not affected by DCVG at any dose level studied (Fig. 2). Urinary excretion of N-acetyl-P-o-glucosaminidase (NAG) was increased 9.5-fold and 10.8-fold that of control rats at 165 and 330 pmolikg of DCVG respectively, whereas hamsters showed a 3.1-fold increase in such excretion only at the highest dose of DCVG (Fig. 3). On the other hand, urinary excretion of NAG in guinea-pigs was not affected by DCVG treatment at any dose level (Fig. 3). Significant increases in levels of urinary proteins were observed in rats due to DCVG treatment at both lower and higher dose levels, whereas in hamsters such increase was seen only at the higher dose of DCVG (Fig. 4). Although guinea-pigs showed an increase in urinary proteins, this was not significant at any dose level, because of large interindividual variations (Fig. 4) in each treated group. Significant increases of 75 and 95% in blood urea nitrogen (BUN) concentration were observed in rats at 165 and 330 pmol/kg of DCVG respectively, whereas in hamsters BUN increased significantly (122%) only at the higher dose (Fig. 5), suggesting a significant decrease in glomerular filtration. A large increase in BUN concentration (320%) was observed in guinea-pigs at the lower dose of DCVG; on the contrary, no such increase in BUN was seen at the higher dose level (Fig. 5). Upon histological examination of kidney slices from rats treated with 165 pmol DCVG, severe necrosis was evident at the outer stripe of the outer medulla (OSOM) with some light necrosis involving cortical proximal tubules (S, and SJ. At the higher

341

4200

0 ‘;;; < v 3

3000

4

2400

FT .f

1800

=

1200

Control 165 qole/kg 330 Fok,‘kg

m m

3600

600

i 0 Rats

Hornsten,

Guinea pigs

Fig. 3. Influence of intraperitoneal treatment with DCVG on the urinary excretion of N-acetyl-B-D-glucosaminidase (NAG) at 24 h. For further explanation, see Fig. 1.

dose, DCVG caused much more severe proximal tubular cell necrosis involving all of the OSOM and the medullary ray. More tubular necrosis was also found to involve the cortical proximal tubules, which appeared to involve both the S, and Sz segments. No histological changes were observed in kidney slices from hamsters treated with the lower dose of DCVG when compared with those in control animals. However, at the higher dose, DCVG produced tubular necrosis throughout the OSOM with few hyaline casts. In DCVG-treated guinea-pigs, extensive proximal tubular necrosis was observed, involving the OSOM at the lower dose level. The cortical proximal tubules involving S, and S2 segments were also damaged with cellular swelling and hyaline eosinophilic casts in the outer medulla as well as swelling of cells at the corticomedullary junction. On the other hand, no such nephrotoxicity was observed when guinea-pigs were treated with a higher dose of DCVG (330 pmolikg), as verified by both biochemical parameters (Figs. l-5) and histological studies. DISCUSSION

The present studies have demonstrated that male Fischer-344 rats are more sensitive to the nephrotoxic action of DCVG than male hamsters which showed no toxicity at the lower dose of 165 pmollkg. An intriguing feature of the toxicity in the rat proximal tubule is that the lesion appears to be focused in the area of the pars recta that contains the proximal straight tubule, or the S, segment, whereas at the higher dose, damage to the pars convoluta, which is composed of the S, and S2 segments, was also observed. On the other hand, the toxicity in the hamster observed at the higher

348

Hamsters

Fig. 4. Influence

of intraperitoneal

treatment further

Guinea pigs

with DCVG on the urinary explanation,

excretion

of proteins

at 24 h. For

see Fig. 1.

dose was restricted to the proximal straight tubule, as verified by histological examination of kidney slices. The present results have further demonstrated that the nature and the extent of renal damage to the proximal tubule induced by DCVG depend not only on the species but also on the dose of DCVG. While a lower dose of DCVG produced severe proximal tubular necrosis in guinea-pigs, a higher dose failed to produce any such renal damage. In other words, guinea-pigs seem to be more susceptible to the nephrotoxic action of DCVG at a lower dose than at a higher dose level, which is surprising and difficult to explain. Renal metabolism of DCVG is required for the expression of its toxicity [5]. The sequential removal of y-glutamyl and glycine moieties, catalyzed by y-GT and a number of dipeptidases respectively, gives rise to S-( 1,2-dichlorovinyl)-L-cysteine (DCVC). The toxicity of DCVG is blocked by inhibitors of y-GT and of dipeptidases, indicating that the GSH conjugate, i.e. DCVG, must be metabolized to the corresponding cysteine conjugate (DCVC) for the expression of toxicity [5]. Moreover, the toxicity of DCVG or DCVC is blocked by amino-oxyacetic acid, an inhibitor of cysteine conjugate P-lyase, indicating the necessity for its further metabolism to a reactive thiol by the pyridoxal-phosphate-dependent /3-lyase [5]. The N-acetylated derivative of DCVC (NAC-DCVC) has been isolated from rats dosed with trichloroethylene [ 171, suggesting DCVC is formed in vivo and subsequently acetylated prior to urinary excretion. It has also been demonstrated that 41.5% of radiolabeled DCVC given to rats was excreted as the N-acetylated form in 24 h [ 181. NAC-DCVC produces a proximal tubular lesion, similar to DCVC in rabbits [19]. Amino-oxy-

349

R&S

Fig. 5. Influence

of intraperitoneal

Hamsten

Guinea pigs

treatment with DCVG on blood urea nitrogen 24 h. For further explanation, see Fig. I.

(BUN) concentrations

at

acetic acid is found to inhibit both covalent binding and toxicity of NAC-DCVC, implying that NAC-DCVC is probably deacetylated to DCVC before being further metabolized [ 193. The present results on species differences in the nephrotoxic response to DCVG cannot be explained solely as due to species differences in renal y-GT activity. Renal y-GT activity is highest in rats, and lowest in guinea-pigs [8]. As such, the nephrotoxicity of DCVG should be highest in rats and lowest in guinea-pigs. While the species differences between rats and hamsters in the nephrotoxic response to DCVG can be explained qualitatively in terms of their differences in renal y-GT activity [8], such an explanation may not be absolutely correct in the case of guinea-pigs. Thus, while renal y-GT activity, which is lowest in guinea-pigs, may be responsible for the absence of any nephrotoxicity in guinea-pigs at the higher dose of DCVG (330 pmoli kg), this is not the case at the lower dose, where guinea-pigs have manifested severe proximal tubular necrosis. On the other hand, species differences in the specific activity of the mercapturic acid (N-acetyl-cysteine-S-conjugate) pathway enzymes may also determine the susceptibility of a given S-conjugate-induced nephrotoxicity. Thus, the guinea-pig forms mercapturic acids much less readily than does the rat or rabbit [9]. Thus, failure to acetylate DCVC to form NAC-DCVC might lead to an increase in such toxicity. If this is true for DCVG at the lower dose, then at the higher dose guinea-pigs should manifest much higher toxicity. On the contrary, just the opposite effect, or no toxic effect, has been observed at the higher dose level. It should be noted, however, that the nephrotoxicity observed in guinea-pigs at a lower dose of DCVG is evidenced both histologically and by increase in BUN con-

350

centrations only, whereas no significant increase was observed in any of the urinary biochemical parameters measured in this study. That is to say, the urinary excretion of y-GT, NAG and glucose did not reflect such toxicity. This is probably due to the severity of toxicity, either due to a decrease in glomerular filtration rates and/or because of an obstruction of the tubular lumen by cellular debris and hyaline casts. A similar observation has been reported previously. Thus, S-( 1,2-dichlorovinyl)-nL-cysteine caused lower urinary glucose excretion at doses which resulted in significantly higher BUN concentrations [5]. In spite of these explanations, why the guinea-pig is relatively insusceptible to toxicity at the relatively higher dose of DCVG still remains a puzzle, and warrants further investigation. ACKNOWLEDGEMENTS

This research was supported by the Medical Research Council of Canada, MA9705. The authors gratefully acknowledge the expert secretarial assistance of Elaine Orphanos.

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