Nephrotoxicity mechanism of 1,1-dichloroethylene in mice

Toxicology Letters ELSEVIER

Toxicology

Nephrotoxicity

Letters 78 (1995) 87-92

mechanism of 1,l -dichloroethylene

in mice

M. Ban*, D. Hettich, N. Huguet, L. Cavelier Service Toxicologic

Industrielle Expkrimentale, Institut National de Recherche et de S&xritk, 54501 Vandoeuvre, France

Received 4 January 1994; revision received 22 September

Avenue de Bourgogne.

1994; accepted 23 September 1994

Male Swiss OF, mice were administered orally with a single dose (200 mg/kg) of l,l-dichloroethylene (DCE). Examination of cryostat kidney sections stained for alkaline phosphatase (APP) revealed damage to about 50% of the proximal tubules at 8 h following DCE administration. Pretreatment with the anionic transport inhibitor probenecid by i.p., (0.75 mmohkg, 30 min prior to and 10 min and 5 h following DCE administration) and with the yglutamyltranspeptidase (GGT) inactivator acivicin by gavage and i.p. (50 mg/kg, 1 h and 30 min prior to DCE administration) failed to prevent DCE-induced renal toxicity. Pretreatment with the /3-lyase inactivator amino-oxyacetic acid (AOAA) by gavage (100 mg/kg, 30 min prior to and 10 min and 5 h following DCE administration), and with the renal cysteine conjugate S-oxidase inhibitor methimaxole by i.p. (40 mg/kg, 30 min prior to DCE administration) reduced the number of damaged tubules by approximately 50 and 60?%1, respectively in mice treated with DCE. The results suggest that the DGE undergoes biotransformation by NADPHcytochrome P450 to several reactive species which conjugate with glutathione (GSH). After arriving in the kidneys, the resulting conjugates reach the renal cells by a mechanism which depends on neither GGT, nor on an anionic transport system which is sensitive to probenecid. Once in the cells, the presumed GSH conjugates and/or their derivatives undergo secondary modification by 8-lyase and cysteine conjugate S-oxidase to reactive metabolite(s). Keywords: 1,lDichloroethylene;

Nephrotoxicity;

Mice; Glutathione

1. Introduction Widely used in industry, for example in the production of plastic film used as food wrappers, or in the treatment of sewage, 1,l -dichloroethylene (DCE) has been known to damage the kidneys, lungs and livers of experimental animals [l-3]. Several studies have been carried out on mice and * Corresponding

author.

rats with regard to DCE-induced covalent binding in these organs [4-81. The results suggested that the pulmonary and hepatic toxicity of DCE requires its bioactivation by a microsomal monooxygenase system into several reactive species which covalently bind to the cellular matrix and presumably induce cell injury. Although the metabolic pathway of DCE-induced toxicity is generally similar in both mice and rats [9], mice were more vulnerable to DCE poisoning than are rats [2].

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M. Ban el al. / ToxicologyLeiters 78 (1994) 87-92

This is partly due to a lower level of pulmonary excretion of unchanged DCE in mice, and to the fact that mice metabolize a greater proportion of the administered DCE than do rats [9,10]. Furthermore, the toxicity of DCE is exacerbated by the depletion of reduced glutathione (GSH) which is involved in its detoxification [ 11,121. Because the GSH is needed for the formation of a conjugate with the reactive metabolite of DCE, mercapturic acid derivates, the products relating to GSH conjugation, were found in the urine of rodents [9-131. The nephrotoxicity of many GSH conjugates has been demonstrated to be dependent on the activity of y-glutamyltranspeptidase (GGT) in the proximal tubules of the kidney [ 14,151. It is possible that the GSH conjugate of DCE found mainly in the kidneys could be transported from the liver since Okine and Gram and Forket et al. [6,7] showed that the reactive metabolites generated in the liver were transported to the lungs and kidneys. N-acetyl-S-carboxymethylcysteine, Although thiodiglycolic acid and methylthioacetylaminoethanol resulting from the association of DCE derivatives with GSH were identified in the urine of animals administered with [ 14C] 1,I-DCE [12,16], the nephrotoxicity mechanism(s) of DCE has not yet been elucidated. In the present study, it was hypothetized that the GSH conjugates of DCE are taken up by the renal membrane-bound enzyme that catalyzes the first step of GSH breakdown, that the transfer of GSH conjugates and/or their derivatives into renal cells is facilited by the anionic transport system which is sensitive to probenecid and that the nephrotoxicity of GSH conjugates and/or their derivatives in the cells is dependent on 2 intracellular renal enzymes: /3lyase and cysteine conjugate S-oxidase. In an attempt to validate these hypotheses, mice received a single dose of DCE (200 mg/kg) by gavage in association with either acivicin, a GGT inhibitor (2 x 50 mg/kg), with probenecid, an anionic transport inhibitor (3 x 0.75 mmol/kg), with amino-oxyacetic acid (AOAA), a /3-lyase inhibitor (3 x 100 mg/kg), or with methimazole, a cysteine conjugate S-oxidase inhibitor (40 mg/kg). These doses were chosen after preliminary studies based on the assessment renal damage.

2. Materials ad methods 1,l -Dichloroethylene (99% pure) was purchased from Merck. Probenecid @-[dipropylsulfamoyl]benzoic acid), acivicin (AT- 125; L-(cr-SSS)-cr-amino3-dichloro-4,5-dihydro-5-isoxazoleacetic acid), AOAA and methimazole (Zmercapto-l-methylimidazole; 1-methylimidazole-2-thiol) were obtained from Sigma. 2. I. Animals Male Swiss OF, mice (IFFA-CREDO), weighing 23-25 g were group-housed for 7 days under controlled environmental conditions and had free access to food (UAR-Alimentation) and water. 2.2. Treatment DCE (200 mg/kg) was administered by oral gavage in corn oil. The interaction of DCE with probenecid, acivicin, AOAA or methimazole was studied in duplicate through 2 series of experiments conducted at the same time of day, in order to control the validity of the intoxication protocol. Each series of experiments involved 4 groups of 10 mice, and nephrotoxicity was assessed 8 h after DCE administration. 2.3. Effect of probenecid on the renal toxicity of DCE Group 1: prober&d (3 x 0.75 mmol/kg) was administered by i.p. injection in isotonic saline (the solution was made alkaline, and then the pH was adjusted to 7.4) 30 min prior to, and 10 min and 5 h following oral administration of DCE (200 mg/kg) in corn oil. Group 2: dosages were the same as those for Group 1, except that alkaline saline solution served as a substitute for probenecid. Group 3: dosages were the same as those for Group 1, but corn oil was substituted for DCE. Group 4: control group in which mice received both the alkaline saline solution and corn oil under the same conditions as above. 2.4. Effect of acivicin (AT-125) on the renal toxicity of DCE Group 1: AT-125 in saline solution (2 x 50 mg/kg) was administered by oral gavage, and by

M. Ban et al. / ToxicologyLetters 78 (1994) 87-92

i.p. injection, respectively 1 h and 30 min prior to administration of DCE. Group 2: dosages were the same as those for Group 1, except that the saline solution served as a substitute for AT-125 Group 3: dosages were the same as those for Group 1, but corn oil was substituted for DCE. Group 4: control group in which mice received the saline solution and corn oil under the same conditions as above. 2.5. Effect of AOAA on the renal toxicity of DCE Group 1: AOAA in distilled water (3 x 100 mg/kg) was administered by oral gavage 30 min prior to and 10 min and 5 h following oral administration of DCE. Group 2: mice were dosed as mice of Group 1 except that the distilled water served as a substitute for AOAA. Group 3: mice were dosed as mice of Group 1, but corn oil was substituted for DCE. Group 4: control group in which mice received the saline solution and corn oil under the same conditions as above. 2.6. Effect of methimazole on the renal toxicity of DCE Group 1: methimazole in saline solution (40 mg/kg) was administered by i.p. injection 30 min prior to oral administration of DCE. Group 2: dosages were the same as those for Group 1, except that the saline solution served as a substitute for methimazole. Group 3: dosages were the same as tRose for Group 1, but corn oil was substituted for DCE. Group 4: control group in which mice received the saline solution and corn oil under the same conditions as above. 2.7. Assessment of renal damage The kidneys were dissected and frozen in liquid nitrogen. Fresh, unfixed cryostat sections of 8 pm were cut and stained for alkaline phosphatase (APP) according to the method defined by Gomori 1171. The sections were incubated for 15 min at 37°C in a freshly prepared medium containing 0.4 mg/ml cr-naphthyl acid phosphate, 1 mg/ml Fast blue RR

89

salt, 20 mg/ml barbitone sodium and 100 mgml magnesium chloride. The final pH of this medium was 9.2. Tubular cross-sections were examined using a light microscope with a IO-fold objective lens and a polygonal field reduced to 0.6 mm diameter. Three hundred renal proximal tubules were examined, with different fields being chosen at random, and the percentage of damaged tubules was calculated. Intact tubular cross-sections were characterized by a large deposit of enzymatic reaction product at the brush-border membrane, while damaged tubules were weakly stained with the reaction product, which is also occasionally found in the cytoplasm in heavy deposits, and dispersed in the lumen of some tubules. The histochemical staining of APP was used in this study for 3 reasons. First, the striking decrease of this enzyme activity, brush border membrane degeneration, was detected in a short time (i.e. 5-6 h) following nephrotoxic chemicals treatment. Second, the coloration of brush border membrane was intense, making differentiation between damaged and undamaged tubules easy. Third, the degrees of renal damage can be quantitatively presented as a percentage of injured tubules. In our previous study, there was a correlation between injured tubule percentage and biochemical parameter, µglobulin [ 181. 2.8. Statistical analysis The interactive renal effect was statistically tested for significance by ANOVA, according to (2 x 2) factorial design. 3. Results None of the mice died during the course of experiments. A single oral administration of DCE at 200 mg/kg evoked nephrotoxicity that is manifested morphologically in proximal tubules. It was found that approximately 50% of the proximal tubules were damaged 8 h after DCE administration. Probenecid, acivicin, AOAA and methimazole did not have any renal effect by themselves. Tables 1 and 2 provide information on the effect of probenecid and AT-125 on the nephrotoxicity of DCE. In the two series of experiments, neither

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M. Ban ei al. / Toxicology Letters 78 (1994) 87-92

Table 1 Effect of probenecid on the nephrotoxicity of DCE in mice. Data are the average values ( l S.D.) for 10 mice in each group

Table 3 Effect of AOAA on the nephrotoxicity of DCE in mice. Data are the average values ( f S.D.) for 10 mice in each group

Treatment

Treatment

Group Group Group Group

1 2 3 4

Percentage of damaged tubules in cryostat kidney sections stained for APP First series of experiments

Second series of experiments

39.83 zt 39.45 f 1.45 * 1.29 l

54.51 * 55.61 + 1.19 zt 1.21 f

3.75 2.71 0.34 0.39

1.73 2.09 0.29 0.26

Group 1: probenecid and DCE, Group 2: alkaline solution and DCE; Group 3: probenecid and corn oil; Group 4: alkaline solution and corn oil.

probenecid nor AT-125 affected the percentage of damaged tubules in DCE-treated mice. Tables 3 and 4 represent the data on the effect of AOAA and methimaxole on the nephrotoxicity of DCE. In the two series of experiments, AOAA pretreatment decreased the percentage of proximal damage induced by DCE by approximately 50%, whereas methimazole did so by approximately 64%. 4. Discussion DCE is metabolized in the liver by NADPHcytochrome P450 to form several reactive species Table 2 Effect of acivicin on the nephrotoxicity of DCE in mice. Data are the average values (i I standard deviation) for IO mice in each group

Group Group Group Group

1 2 3 4

Group Group Group Group

1 2 3 4

Second series of experiments

44.78 f 43.90 f 1.23 f 1.11 l

36.99 zt 43.21 f 1.45 f 1.26 f

2.39 2.13 0.36 0.33

2.89 4.08 0.28 0.57

Group 1: acivicin and DCE; Group 2: saline solution and DCE; Group 3: acivicin and corn oil; Group 4: saline solution and corn oil.

Second series of experiments

26.02 zt 56.38 f 1.06 l 1.30 f

27.21 zt 52.19 zt 1.37 zt 1.29 f

5.50’ 1.11 0.30 0.20

3.83* 1.41 0.26 0.35

that conjugate with GSH via glutathione Stransferase, or by another enzyme such as alcohol dehydrogenase [5- 191. However, although the nephrotoxicity of DCE may be due to the translocation of reactive metabolites from the liver to the kidney [6], the kidney is a possible site for the metabolism of DCE [20]. It is possible that the DCE metabolites are transferred directly to the kidney (liver - blood - kidney) or indirectly by enterohepatic recirculation since biliary metabolites of DCE account for 30-40% of the DCE metabolite excreted in urine [9,16]. Table 4 Effect of methimaxole on the nephrotoxicity of DCE in mice. Data are the average values (f I standard deviation) for 10 mice in each group

Percentage of damaged tubules in cryostat kidney sections stained for APP First series of experiments

First series of experiments

Group 1: AOAA and DCE; Group 2: distilled water and DCE; Group 3: AOAA and corn oil; Group 4: distilled water and corn oil. *Significantly different from Group 2 (P < 0.01).

Treatment Treatment

Percentage of damaged tubules in cryostat kidney sections stained for APP

Group Group Group Group

1 2 3 4

Percentage of damaged tubules in cryostat kidney sections stained for APP First series of experiments

Second series of experiments

19.17 t 53.66 f 0.78 f 0.65 t

21.46 f 57.08 zt 1.35 l 1.42 *

6.11. 2.83 0.28 0.20

5.03’ 4.36 0.35 0.36

Group I: methimaxole and DCE; Group 2: saline solution and DCE; Group 3: methimaxole and corn oil; Group 4: saline solution and corn oil. lSignificantly different from Group 2 (P < 0.01).

M. Ban et al. / Toxicology Letters 78 (1994) 87-92

In our previous studies on the nephrotoxicity of hexachloro-1,fbutadiene and methyl mercury in mice, we reported that the major route of metabolite detoxification was by conjugation with GSH, and subsequently by uptake of the GSH conjugates into renal cells via GGT and anionic transport system [14,21]. Therefore, GGT has been shown to be essential for the activation of GSH conjugates and for the resulting nephrotoxicity

91

the renal cells by a mechanism which depends on neither GGT nor an anionic transport system which is sensitive to probenecid. Once in the cells, the presumed GSH conjugates and/or their metabolites undergo secondary modification by B-lyase and cysteine conjugate S-oxidase to reactive metabolite(s). Acknowledgements

[la Contrarily, in this study it was found that the GSH conjugates of DCE were not broken down by the renal-bound enzyme, GGT, and not transported into renal cells via anionic transport system. Neither AT-125, which inhibits GGT activity, nor probenecid, which inhibits anionic transport, prevented the nephrotoxicity caused by DCE. It still remains unclear how the GSH conjugates are degraded and taken up by renal cells. Some explanations could be proposed: (1) the GSH conjugates of DCE are not the substrate for GGT, they are degraded by other enzyme such as aminopeptidase; (2) AT-125 does not efficiently inhibit the entire activity of GGT in the bile, intestine and kidney; (3) the GSH conjugates of DCE or their derivatives are transported into the renal cells by passive diffusion after membrane fixation. However, in this study we found that upon reaching the cell, the GSH conjugates of DCE or their derivatives undergo a secondary transformation at least by 2 renal cell enzymes: fl-lyase and cysteine conjugate S-oxidase. The first of these, plyase, plays an important role in cysteine Sconjugate degradation of glutathione conjugates [14,22]. The case of the cysteine conjugate Soxidase, which is involved in the bioactivation mechanism of cis-platinum II diammine dichloride, was recently reported in our laboratory [23]. Pretreatment with @-lyaseand S-oxidase inhibitors reduced the percentage of injured tubules in mice treated with DCE by approximately 50 and 60%, respectively. In summary, the findings described here point to the possibility that DCE undergoes biotransformation by NADPH-cytochrome P450 to several reactive species which conjugate with GSH. The resulting conjugates may be transferred to bile or blood circulation. After arriving in the kidneys, the GSH conjugates and/or their metabolites reach

The authors wish to thank secretarial work.

C. Sellier for

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[I81 Payan, J.P., Saillenfait, A.M., Jkydon, D., Ban, M. and de Ceaurrix, J. (1990) Pregnancy-associated changes in renal toxicity of cadmium-metallothionein: possible role of intracellular metallothionein. Toxicology 65,223-232. [19] Costa, A.K. and Ivanetich, K.M. (1982) Vinylidene chloride: its metabolism by hepatic microsomal cytochrome P450 in vitro. B&hem. Ph armacol. 31, 2083-2092. [20] Masuda, Y. and Nakayama, N. (1983) Protective action of diethyldithiocarbamate and carbon disulfide against acute toxicities induced by I,l-dichloroethylene in mice. Toxicol. Appl. Pharmacol. 71, 42-53. [21] Ban, M. and de Ceaurriz, J. (1988) Probenecid-induced protection against acute hexachloro-1,3-butadiene and methyl mercury toxicity of the mouse kidney. Toxicol. Lett. 40, 71-76. 1221Dekant, W., Vamvakas, S. and Anders, M.W. (1989) Bicactivation of nephrotoxic haloalkenes by glutathione conjugation: formation of toxic and mutagenic intermediates by cysteine conjugate f3-lyase. Drug Metab. Rev. 20, 43-83. [23] Ban, M., Hettich, D. and Huguet, N. (1994) Nephrotoxicity mechanism of c&platinum (II) diammine dichloride in mice. Toxicol. L.&t. 71, 161-168.