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Dichlorovinyl cysteine (DCVC)-induced enzymuria in mice: potential application in occupational toxicology?
139
Toxicology Letfers, 53 (1990) 139-141 Elsevier
TOXLET
02400
Dichlorovinyl cysteine (DCVC)-induced enzymuria in mice: potential application in occupational toxicology?
Gull-Britt Hagberg, Ulrika Nordlander
and Per Ola Darnerud
Department of Toxicology, Uppsala University, Uppsala (Sweden)
Key worak Halogenated Alkaline
phosphatase;
alkenes; Cysteine Lactate
conjugates;
Nephrotoxicity;
y-Glutamyl
transpeptidase;
dehydrogenase
Since the nephrotoxic effects of the cysteine conjugate of trichloroethylene, dichlorovinyl cysteine (DCVC), was discovered, a number of additional toxic conjugates of halogenated alkenes have been found [e.g. Ref. 11. Using DCVC as model compound, studies on its accumulation in mice, and the conditions for renal binding and toxicity, have been performed [2,3]. In order to validate a non-invasive method of monitoring the DCVC-induced kidney lesion, selected renal enzymes were quantified in the urine of treated mice. The results were compared with those from blood urea nitrogen (BUN) measurements. Clinical applications of the results are discussed. Commercial kits for the determination of y-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) (if necessary modified to fit urinary measurements), and of BUN, were used (all from Sigma Chem. Co., St. Louis, MO, U.S.A.). Mice (female C57BL) were treated with DCVC in groups of 4 (see Table I). Twenty-four hours after the oral administration, the urine was collected, centrifuged, and kept at 4°C until analysis. The animals were killed and the kidneys were taken out for histopathological examination (paraffin sections, H-E staining). The results from the urinary enzyme (total activity/24 h + SD) and BUN measurements on DCVC-treated mice are presented in Table I. The GGT and LDH values
Address for correspondence: BOX 594, S-751 24 Uppsala,
0378-4274/90/S
P.O.
Damerud,
Department
of Toxicology,
Uppsala
Sweden
3.50 @ 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
University,
BMC,
140 TABLE I MOUSE URINARY ENZYME (pGLUTAMYL TRANSPEPTIDASE, ALKALINE PHOSPHATASE, LACTATE DEHYDROGENASE; TOTAL ACTIVITY/24 h) AND BLOOD UREA NITROGEN LEVELS 24 h AFTER ORAL DCVC TREATMENT Treatment
Control 0.1 mg/kg 0.5 mg/kg
1w/kg 5 mg/kg 10 mdkg 25 mg/kg
GGT (Sigma U)
ALP (Sigma U)
LDH (B-B U)
BUN (mddl)
42k
4
0.6kO.2
46+
58
91*
44
1.9+ 1.4
35&
35
14*
109*
34
0.8kO.l
22&
32
_
289+ 163 3408+ 1653 3791+ 1767 135Ok 1650
0.7kO.4 1.6kO.6 2.3kO.2 10.2k5.2
76+ 60 363051127 42405 729 5127k 1120
13+ 2 2
14, 1 31+ 3 58& 5 95+47
Mean values (n =4) f SD.
were markedly elevated from 1 to 5 mg/kg DCVC, whereas ALP gave a less pronounced increase with dose. At 5 mg/kg the enzyme levels for GGT and LDH were increased above control by factors of 81 and 79, respectively. At the highest dose the GGT values returned to lower levels, whereas the LDH levels were moderately increased. The BUN measurements showed a better dose-response correlation (from 1 mg/kg), showing highest values in the highest dose group. The histopathological results at higher DCVC doses (5-25 mg/kg) are in accord with those presented earlier in mice [2]. The 1 mg/kg dose gave a visible alteration of the tubular morphology (dilatation of tubules, some vacuoles in the epithelium but mostly intact brush border); the 0.1 mg/kg dose, however, gave hardly any visible effects (some dilatation of tubules). The results show that the nephrotoxic response to DCVC can be detected by use of enzymuria measurements. This method has earlier been used in nephrotoxicity studies on another cysteine conjugate, pentachlorobutadienyl cysteine (PCBC) [4]. Especially GGT and LDH are markedly increased after DCVC treatment, and the elevation at 5 mg/kg coincides with the development of a necrosis of the straight proximal tubules (SPT). As GGT has the highest activity in the brush-border membrane of the SPT region [2,5], and as DCVC initially hits the very same segment, the high elevation of GGT is not surprising. In the case of ALP, the difference in tubular distribution [S] as compared to GGT, could not likely explain the low urinary activities after DCVC treatment. The LDH activity, reflecting cytosolic leakage of enzymes, rises steeply at 5 mg/kg, similar to GGT, and probably mirrors the onset of cell death in the SPT region. However, the fact that the activities of GGT and LDH, in contrast to the BUN levels, does not increase significantly with increasing DCVC doses above 5 mg/kg, is not compatible with the histopathological results which show a more extensive lesion after 25 mg/kg than after 5 mg/kg [2]. Possible reasons for this dis-
141
crepancy at higher DCVC doses may include: (1) enzyme trapping and tubular occlusion, (2) enzyme inactivation, and (3) sediment binding of enzymes. Clinical enzymuria measurements may be useful as a non-invasive method of monitoring nephrotoxic effects of halogenated alkenes. Thus, the urinary measurement of GGT and LDH seems a sensitive marker of kidney lesion development, although quantitative data on the kidney lesion could not be obtained. As a dose-dependent marker of kidney lesions, although less sensitive, BUN measurements could supplement the enzyme analyses. In the occupational situation, the mode of exposure is quite different from that in the present experiment. The effect of a continuous, lowdose exposure of these chemicals on the appearance of urinary enzymes should therefore be the aim of future studies. REFERENCES 1 Anders, M.W., Lash, L.H., Dekant, W., Elfarra, A.A. and Dohn, D.R. (1988) Biosynthesis and metabolism of glutathion-S-conjugates to toxic forms. CRC Crit. Rev. Toxicol. 18, 311-341. 2 Damerud, P.O., Brandt, I., Feil, W.J. and Bakke, J.E. (1988) S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) in the mouse kidney: correlation between tissue-binding and toxicity. Toxicol. Appl. Pharmacol. 95, 4233434. 3 Darnerud, P.O., Brand& I., Feil, V.J. and Bakke, J.E. (1989) Dichlorovinyl cysteine (DCVC) in the mouse kidney: tissue-binding and toxicity after glutathione depletion and probenecid treatment. Arch. Toxicol. 63, 345350. 4 Nash, J.A., King, L.J., Lock, E.A. and Green, T. (1984) The metabolism and disposition of hexachloro1,3-butadiene in the rat and its relevance to nephrotoxicity. Toxicol. Appl. Pharmacol. 73, 124137. 5 Guder, W.G. and Ross, B.D. (1984) Enzyme distribution along the nephron. Kidney Int. 26, 101-l 11.
Toxicology Letfers, 53 (1990) 139-141 Elsevier
TOXLET
02400
Dichlorovinyl cysteine (DCVC)-induced enzymuria in mice: potential application in occupational toxicology?
Gull-Britt Hagberg, Ulrika Nordlander
and Per Ola Darnerud
Department of Toxicology, Uppsala University, Uppsala (Sweden)
Key worak Halogenated Alkaline
phosphatase;
alkenes; Cysteine Lactate
conjugates;
Nephrotoxicity;
y-Glutamyl
transpeptidase;
dehydrogenase
Since the nephrotoxic effects of the cysteine conjugate of trichloroethylene, dichlorovinyl cysteine (DCVC), was discovered, a number of additional toxic conjugates of halogenated alkenes have been found [e.g. Ref. 11. Using DCVC as model compound, studies on its accumulation in mice, and the conditions for renal binding and toxicity, have been performed [2,3]. In order to validate a non-invasive method of monitoring the DCVC-induced kidney lesion, selected renal enzymes were quantified in the urine of treated mice. The results were compared with those from blood urea nitrogen (BUN) measurements. Clinical applications of the results are discussed. Commercial kits for the determination of y-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) (if necessary modified to fit urinary measurements), and of BUN, were used (all from Sigma Chem. Co., St. Louis, MO, U.S.A.). Mice (female C57BL) were treated with DCVC in groups of 4 (see Table I). Twenty-four hours after the oral administration, the urine was collected, centrifuged, and kept at 4°C until analysis. The animals were killed and the kidneys were taken out for histopathological examination (paraffin sections, H-E staining). The results from the urinary enzyme (total activity/24 h + SD) and BUN measurements on DCVC-treated mice are presented in Table I. The GGT and LDH values
Address for correspondence: BOX 594, S-751 24 Uppsala,
0378-4274/90/S
P.O.
Damerud,
Department
of Toxicology,
Uppsala
Sweden
3.50 @ 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
University,
BMC,
140 TABLE I MOUSE URINARY ENZYME (pGLUTAMYL TRANSPEPTIDASE, ALKALINE PHOSPHATASE, LACTATE DEHYDROGENASE; TOTAL ACTIVITY/24 h) AND BLOOD UREA NITROGEN LEVELS 24 h AFTER ORAL DCVC TREATMENT Treatment
Control 0.1 mg/kg 0.5 mg/kg
1w/kg 5 mg/kg 10 mdkg 25 mg/kg
GGT (Sigma U)
ALP (Sigma U)
LDH (B-B U)
BUN (mddl)
42k
4
0.6kO.2
46+
58
91*
44
1.9+ 1.4
35&
35
14*
109*
34
0.8kO.l
22&
32
_
289+ 163 3408+ 1653 3791+ 1767 135Ok 1650
0.7kO.4 1.6kO.6 2.3kO.2 10.2k5.2
76+ 60 363051127 42405 729 5127k 1120
13+ 2 2
14, 1 31+ 3 58& 5 95+47
Mean values (n =4) f SD.
were markedly elevated from 1 to 5 mg/kg DCVC, whereas ALP gave a less pronounced increase with dose. At 5 mg/kg the enzyme levels for GGT and LDH were increased above control by factors of 81 and 79, respectively. At the highest dose the GGT values returned to lower levels, whereas the LDH levels were moderately increased. The BUN measurements showed a better dose-response correlation (from 1 mg/kg), showing highest values in the highest dose group. The histopathological results at higher DCVC doses (5-25 mg/kg) are in accord with those presented earlier in mice [2]. The 1 mg/kg dose gave a visible alteration of the tubular morphology (dilatation of tubules, some vacuoles in the epithelium but mostly intact brush border); the 0.1 mg/kg dose, however, gave hardly any visible effects (some dilatation of tubules). The results show that the nephrotoxic response to DCVC can be detected by use of enzymuria measurements. This method has earlier been used in nephrotoxicity studies on another cysteine conjugate, pentachlorobutadienyl cysteine (PCBC) [4]. Especially GGT and LDH are markedly increased after DCVC treatment, and the elevation at 5 mg/kg coincides with the development of a necrosis of the straight proximal tubules (SPT). As GGT has the highest activity in the brush-border membrane of the SPT region [2,5], and as DCVC initially hits the very same segment, the high elevation of GGT is not surprising. In the case of ALP, the difference in tubular distribution [S] as compared to GGT, could not likely explain the low urinary activities after DCVC treatment. The LDH activity, reflecting cytosolic leakage of enzymes, rises steeply at 5 mg/kg, similar to GGT, and probably mirrors the onset of cell death in the SPT region. However, the fact that the activities of GGT and LDH, in contrast to the BUN levels, does not increase significantly with increasing DCVC doses above 5 mg/kg, is not compatible with the histopathological results which show a more extensive lesion after 25 mg/kg than after 5 mg/kg [2]. Possible reasons for this dis-
141
crepancy at higher DCVC doses may include: (1) enzyme trapping and tubular occlusion, (2) enzyme inactivation, and (3) sediment binding of enzymes. Clinical enzymuria measurements may be useful as a non-invasive method of monitoring nephrotoxic effects of halogenated alkenes. Thus, the urinary measurement of GGT and LDH seems a sensitive marker of kidney lesion development, although quantitative data on the kidney lesion could not be obtained. As a dose-dependent marker of kidney lesions, although less sensitive, BUN measurements could supplement the enzyme analyses. In the occupational situation, the mode of exposure is quite different from that in the present experiment. The effect of a continuous, lowdose exposure of these chemicals on the appearance of urinary enzymes should therefore be the aim of future studies. REFERENCES 1 Anders, M.W., Lash, L.H., Dekant, W., Elfarra, A.A. and Dohn, D.R. (1988) Biosynthesis and metabolism of glutathion-S-conjugates to toxic forms. CRC Crit. Rev. Toxicol. 18, 311-341. 2 Damerud, P.O., Brandt, I., Feil, W.J. and Bakke, J.E. (1988) S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) in the mouse kidney: correlation between tissue-binding and toxicity. Toxicol. Appl. Pharmacol. 95, 4233434. 3 Darnerud, P.O., Brand& I., Feil, V.J. and Bakke, J.E. (1989) Dichlorovinyl cysteine (DCVC) in the mouse kidney: tissue-binding and toxicity after glutathione depletion and probenecid treatment. Arch. Toxicol. 63, 345350. 4 Nash, J.A., King, L.J., Lock, E.A. and Green, T. (1984) The metabolism and disposition of hexachloro1,3-butadiene in the rat and its relevance to nephrotoxicity. Toxicol. Appl. Pharmacol. 73, 124137. 5 Guder, W.G. and Ross, B.D. (1984) Enzyme distribution along the nephron. Kidney Int. 26, 101-l 11.