Toxicology, 64 (1990) 113--127 Elsevier Scientific Publishers Ireland Ltd.
The interactions of cis-diamminedichloroplatinum with metallothionein and glutathione in rat liver and kidney Catherine A.M. Suzuki* and M.G. Cherian** Departments of Pharmacology and Toxicology and Pathology, University of Western Ontario, London, Ontario N6A 5C1 (Canada) (Received January 23rd, 1990; accepted May 1lth, 1990)
Summary The involvement of metallothionein (MT) in the nephrotoxicity of cis-diamminedichloroplatinum (c-DDP) was investigated in rats using enzyme excretion and histology as indicators of renal damage. In addition, the effects of renal glutathione (GSH) depletion on the nephrotoxicity of c-DDP was assessed by organic anion transport in renal cortical slices. A dose of 6.0 mg c-DDP/kg body wt, i.p. was administered to rats either as a single injection of 6.0 mg/kg or as six daily injections of 1.0 mg/kg. Concentrations of platinum (Pt) after c-DDP injection in both dosing regimens were approximately 12 i~g/g in kidney and 2 pg/g in liver. However, there were no increases in either hepatic or renal concentrations of MT after both series of c-DDP injections. Fractionation of kidney cytosols from c-DDP injected rats on Sephadex G-75 columns revealed that 60--70070 of cytosolic Pt was associated with proteins of high molecular weight and 15--20070 of the Pt associated with the low molecular weight ligands. No discernable Pt peak was detected in the elution volume of MT. Pretreatment of rats with ZnSO~ increased both hepatic and renal concentrations of MT, but there was no Pt associated with the MT fraction after a subsequent injection of cDDP. Small increases in the urinary excretion of the lysosomal enzyme, N-acetyl-p-D-glucosaminidase and two brush border enzymes, alkaline phosphatase and y-glutamyltranspeptidase were observed 2 and 3 days after a single injection of c-DDP (6.0 mg/kg body wt, i.p.). Urinary creatinine excretion decreased by 50°70 1 day after c-DDP injection and continued to decrease for the next 2 days. On the third day after c-DDP treatment, a small but significant decrease in body weight was also observed in the c-DDP injected animals. Pretreatment with Zn did not alter the c-DDP-induced enzymuria or renal tubular damage but slightly attenuated both the decrease in creatinine excretion and the loss in body weight. Uptake of the organic anion, p-aminohippuric acid (PAH) was reduced at 12 and 24 h after cDDP injection. Reduction of tissue GSH concentrations by pretreatment with buthionine sulfoxime (BSO), resulted in only a slight increase in the c-DDP-induced inhibition of PAH uptake at 24 h after c-DDP injection.
*Present Address: Drug Toxicology Division, Health Protection Branch, Sir F.G. Banting Research Centre, Tunney's Pasture, Ottawa, Ontario, Canada. K1A OL2 **To whom all correspondence should be addressed. 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
113
These results suggest that, in rats, neither MT nor GSH appear to play major roles in the binding or nephrotoxicityof c-DDP. Key words: C/s-diamminedichloroplatinum;Metallothionein; Glutathione; Nephrotoxicity Introduction
Cis-diamminedichloroplatinum (c-DDP) is a member of a group of antineoplastic agents containing the divalent metal, platinum (Pt). Although the use of cDDP often results in the development of deleterious side effects, the use of this potent chemotherapeutic agent is primarily limited by a dose-related nephrotoxicity [1--3]. Since the renal damage caused by c-DDP treatment is similar to some of the toxic effects of metals, it has been hypothesized that the renal damage induced by c-DDP may be mediated by the Pt moiety itself [4]. As a result, it has been suggested that the high cysteine-containing intracellular protein, metallothionein (MT) which is known to play an important role in the detoxification of cadmium (Cd) [5--7], may also reduce the nephrotoxicity of c-DDP. The induction of MT synthesis and binding of Pt to MT following c-DDP injection into experimental animals has been reported by a number of investigators [8--10] and consequently the induction of MT as a protective mechanism against the nephrotoxicity of c-DDP has been investigated [11--14]. However, in a study by Mason et al. [15], following c-DDP injection to rats, there was no induction of MT synthesis or any binding of the Pt with pre-existing MT. Thus, the involvement of MT in the metabolism and the renal toxicity of c-DDP remains unclear and was further investigated in the present study. Glutathione (GSH), the major non-protein thiol source in the cell, plays an important role in the metabolism and detoxifieation of a number of chemicals and its potential role in metal detoxification has been recently investigated [16-18]. It has been hypothesized that the high levels of GSH in kidney and liver may provide a readily accessible pool of thiol binding sites for metal binding prior to the synthesis of MT. Since GSH has been implicated in the metabolism of c-DDP [19], the effects of tissue GSH depletion on the toxicity of c-DDP was studied. Methods
M T induction and Pt binding Male Sprague--Dawley rats (200--250 g) (Charles River, Canada) used in this study were maintained on a 12 h light and dark cycle, fed standard Purina rat chow and given water ad libitum. All the c-DDP (Bristol Myers, Syracuse, N.Y.) solutions were freshly prepared just prior to injection. A dose of 6.0 mg c - D D P / kg body wt, i.p. was chosen for the present study since a similar dose was used in a previous report to demonstrate MT induction [8]. However, since this dose has also been shown to result in pathological changes to the kidney [20], the same total dose was injected into rats as 6 injections of 1.0 mg c-DDP/kg body wt, i.p. over 6 days in an attempt to reduce the immediate toxic effects of c-DDP.
114
Animals were killed under pentobarbital anaesthesia by exsanguination from the abdominal aorta at 24 h after the last injection of c-DDP and livers and kidneys were removed and analyzed for Pt content by flameless atomic absorption spectrometry (FAAS). Tissue samples (approx. 0.5 g) were digested for 24 h in nitric acid (15 N) and the resultant solution was evaporated to near dryness by gentle heating. The residue was resuspended in 1 N HC1, again evaporated by heating, the residue resuspended in 0.1 N HCI and again evaporated to near dryness. The sample was diluted to a final volume of 5.0 ml with 0.1 N HCI and Pt concentrations in the samples were measured by FAAS on a Varian Spectra 30 (Varian Canada, Georgetown, Ontario). The parameters for Pt analysis by FAAS were as follows: Lamp current, 7.0 mAmps; slit width, 0.2 nm; wavelength, 265.9 nm; background correction on. Tissue MT concentrations were determined using the silver saturation method [21]. To examine the protein binding profile of Pt in the cytosol, immediately after the animal was killed, the kidney was removed and a 0.5 g sample of the kidney was homogenized in 4 vols. of a 250 mM sucrose-Tris (50 mM)-KC1 (25 mM)MgCI 2 (5 raM) (TKM) buffer, pH 8.6 and the homogenate was centrifuged at 105 000 g for 1 h. An aliquot of the resultant supernatant (0.3 ml) was fractionated on a standardized Sephadex G-75 column (0.9 cm x 60.0 cm) and eluted with 10 mM TrismHCl buffer (pH 8.6). The fractions (1.0 ml) were analyzed for Pt by FAAS. To examine the in vivo binding of Pt to MT, rats were pretreated with ZnSO 4 (20 mg Zn/kg body wt, s.c.) 24 h prior to a single c-DDP injection (6 mg/kg body wt, i.p.). Twenty-four hours after c-DDP injection, animals were killed by exsanguination and tissues (liver and kidney) were analyzed for Pt, Zn and MT. Kidney cytosols were fractionated on standardized Sephadex G-75 columns and fractions were analyzed for Pt and Zn as previously described.
Urinary enzyme excretion The measurement of urinary enzymes has been used as a sensitive, non-invasive method for assessing renal damage of a number of nephrotoxicants [22,23] and it has been suggested that urinary enzymes may provide a sensitive indicator of c-DDP-induced renal damage [24]. Since injected c-DDP is excreted primarily in urine, it is important to determine the effects of c-DDP on enzyme activity in urine. The direct in vitro effect of c-DDP on the activities of the enzymes, alkaline phosphatase (ALP), r-glutamyltranspeptidase (GGT) and N-acetyl-/3-D-glucosaminidase (NAG) in urine was examined by incubating 2-ml aliquots of urine for 2 h at 4°C with and without addition of c-DDP in concentrations of 10--100 /~g Pt/ml of urine. Male Sprague--Dawley rats (250--270 g) were housed individually in plastic Nalgene metabolic cages. For 3 days prior to treatment, 24 h urine samples were collected over ice and analyzed for creatinine, ALP, GGT and NAG as previously described [25]. These values represented control urinary enzyme excretion levels. After a single injection of c-DDP (6.0 mg/kg body wt, i.p.), 24-h urine samples were collected for another 3 days and urinary enzyme levels and Pt were 115
measured. Enzyme excretion values were calculated as the amount excreted per 24-h period and expressed as a percentage of control. Rats were killed at the end of 3 days and body weight and the renal concentrations of Pt, Zn and MT were measured. A section of kidney was fixed in neutral 10% formalin, embedded in paraffin, and stained with haematoxylin and eosin (H&E) for examination by light microscopy. A second group of rats were injected with ZnSO 4 (20 mg Zn/kg body wt, s.c.) 24 h before the single c-DDP injection (6.0 mg/kg body wt, i.p.) and urinary enzyme excretion, kidney morphology and tissue Pt, Zn and MT concentrations were analyzed as described before. GSH Depletion Tissue GSH concentrations were decreased in male Sprague--Dawley rats (200 --250 g), by treatment with a specific inhibitor of GSH biosynthesis, buthionine sulfoxime (BSO) (Sigma Chemical Co., St. Louis, MO.) (4 mmol/kg body wt, s.c.). Four hours after BSO injection, rats were injected with c-DDP (a single injection of 6.0 mg/kg body wt, i.p.) and killed by decapitation 4, 12 or 24 h later. Kidney and liver concentrations of Pt, GSH and MT were measured. Rats were killed at the same time of day since tissue GSH content is subject to diurnal variations [26]. Damage to the kidney was assessed by measuring the uptake of paminohippuric acid (PAH) in renal slices as previously described [27l. Kidney samples were homogenized and cytosols were prepared for fractionation on a Sephadex G-75 column and column fractions were analyzed for Pt and Zn as described before. Statistical analysis Statistical differences were calculated with Student's t-test or one-way analysis of variance (ANOVA) where required. Multiple comparison testing was performed using the Student-Neuman-Keuls test. Differences were considered statistically significant if they achieved a significance level of P < 0.05. Results
M T induction and Pt binding After the injection of rats with c-DDP (6.0 mg/kg) by either dosing regimen, Pt concentrations in tissues were approximately 10--13 /ag/g in kidney and 2--3 /ag/g in liver (Table I). The concentrations of Zn and MT in these tissues were similar to controls (Table I). Kidney cytosols from individual rats injected with c-DDP were fractionated on a Sephadex G-75 column. A typical elution profile is shown in Fig. 1 for Pt in a kidney cytosol of a rat which had been injected with c-DDP at a dose of 1.0 mg/ kg each day for 6 days. Approximately 60--70% of cytosolic Pt was associated with proteins of high molecular weight (HMW) with 15--20% associated with the
116
TABLE 1 RENAL AND HEPATIC CONCENTRATIONS OF Pt, Zn AND MT IN RATS AFTER c-DDP INJECTION WITH OR WITHOUT Zn PRETREATMENT All animals except controls were injected with c-DDP and killed 24 h after the last injection. The numbers in parentheses represent the number of injections and the dose of c-DDP of each injection. Zn-pretreated rats were injected with ZnSO, (20 mg Zn/kg body wt.) 24 h prior to c-DDP injection. Values represent the mean ± S.D., n = 6. Treatment
Pt (/ag/g tissue)
Zn ~g/g tissue)
MT (~g/g tissue)
Control
Kidney Liver
N.D. N.D.
26.79 ± 5.24 30.54 ± 2.68
32.60 ± 8.85 ±
5.66 1.96
c-DDP (6x 1 mg/kg)
Kidney
10.02 ± 2.31
33.79 ± 6.07
29.27 ±
9.41
2.45 ± 0.09
31.65 ± 2.49
7.74 -4- 1.36
13.00 ± 0.77
26.63 ± 3.97
35.32 _+ 6.41
3.36 ± 0.35
33.92 ± 3.73
15.52 ±
10.81 _+ 1.52
28.99 ± 1.65
86.55 ± 17.82"
2.91 ± 0.44
41.07 ± 4.71"
150.77 _+ 15.41"
Liver
c-DDP 0×6 mg/kg)
Kidney
Zn + c-DDP (6x 1 mg/kg)
Kidney
Liver
Liver
5.22
N.D. Not determined.
*Significantly different from control (P < 0.05).
low m o l e c u l a r weight ( L M W ) ligands. T h e r e m a i n d e r o f the P t was d i s t r i b u t e d b e t w e e n the H M W a n d L M W regions with n o a p p a r e n t p e a k in the region where M T is eluted (10,000 d a l t o n s ) . T h e cytosolic d i s t r i b u t i o n o f P t a f t e r the single high d o s e (6.0 m g / k g ) i n j e c t i o n o f c - D D P was similar ( d a t a n o t shown). Recoveries o f P t in the f r a c t i o n s were b e t w e e n 92--100%0 o f the t o t a l a m o u n t o f P t a p p l i e d to the c o l u m n s . P r e t r e a t m e n t with Z n at 24 h p r i o r to c - D D P i n j e c t i o n resulted in significant increases in b o t h renal a n d h e p a t i c M T c o n c e n t r a t i o n s as c o m p a r e d to the c - D D P a l o n e injected g r o u p ( T a b l e I). T h e a c c u m u l a t i o n o f P t in liver a n d k i d n e y was n o t altered b y Z n p r e t r e a t m e n t . T h e gel f i l t r a t i o n o f cytosols f r o m kidneys o f a c - D D P injected rat p r e t r e a t e d with Z n r e v e a l e d t h a t a l t h o u g h a p p r o x i m a t e l y 15% o f cytosolic Z n was e l u t e d in the r e g i o n o f M T , there was no m e a s u r a b l e a m o u n t o f P t a s s o c i a t e d with this p e a k ( d a t a n o t s h o w n ) . S i m i l a r to the results in Fig. 1, the m a j o r i t y o f t h e P t ( 6 0 % ) was f o u n d with the H M W p r o t e i n s with a n o t h e r 2 2 % o f the P t a s s o c i a t e d with the L M W l i g a n d s a n d the r e m a i n i n g P t d i s t r i b u t e d f a i r l y u n i f o r m l y b e t w e e n the H M W a n d L M W f r a c t i o n s ( d a t a not shown).
117
oo
17.5 40.0 28.5 28.0
-+ +. +. ---
3.8 11.8 a 4.21 13.7
N.D. 433.5 --4-- 55.9 34.0 _+ 3.7 19.2 +. 4.1
10.9 5.6 3.3 4.9
-+ -+ +. +.
0.8 1.6 "b 0.8 a'b 1.9~
15.8 41.2 25.8 25.3
+- 2.9 +. 12.5 ° -+ 8.9 ~ +. 6.9 ~
Volume (mi/day)
Creatinine (mg/day)
Volume (ml/day)
Pt (~g/day)
Zn + c-DDP
c-DDP
Treatment
N.D. Not Determined. •Significantly different from control (P < 0.05). bSignificantly different from Zn + c-DDP group.
Control 1 2 3
Day After c-DDP Injection
N.D. 480.5 +. 39.5 42.7 +. 9.0 23.8 +. 3.5
Pt Og/day)
9.7 7.5 5.9 4.5
+++. +.
1.2 1.6 1.3 I 0.8 a
Creatinine (rag/day)
Rats were injected with c-DDP (6.0 m g / k g body wt.) and killed 3 days later. Zn-pretreated animals were injected with ZnSO 4 (20 mg Z n / k g body wt.) 24 h prior to c-DDP injection. Controls are values before c-DDP injection. Values represent the mean +. S.D., n = 6.
DAILY URINE VOLUMES AND U R I N A R Y E X C R E T I O N OF Pt AND CREATININE F O L L O W I N G c-DDP INJECTION
TABLE II
\
~ ,o l
~
so 2o
I 20
IO
30
40
FRACTION NUMBER Fig. 1. The cytosolic distribution patterns of Pt in kidney after the injection of c-DDP (1 mg/kg body wt) daily for 6 days. Twenty-four hours after the last injection, rats were sacrificed, kidneys removed and homogenized in a 250 mM sucrose-TKM buffer (pH 8.6), centrifuged at 105 000 g and an aliquot of the resultant supernatant applied on a Sephadex G-75 column. Fractions were analyzed for Pt by FAAS. 300
A
I
.
I
. GGT
=
NAG
COOP
0
I
2
3
4
!4
/
g ZnSO 4
| i f COOP
I 2
I 3
I 4
TIME (Doys) Fig. 2. Urinary excretion profiles of GGT (A), ALP ( , ) and NAG (O) after a single injection of cDDP (6.0 mg/kg body wt). (A) Saline pretreated. (B) ZnSO4 (20 mg Zn/kg body wt) pretreated 24 h prior to c-DDP injection. Twenty-four hour urine samples were collected over ice and analyzed for ALP, GGT and NAG activities. Activities of the enzymes were expressed as percentages of control values. (Means ± S.D., n = 6) *Significantly different from control (P < 0.05).
119
Urinary enzyme excretion Within the first 24-h period after c-DDP injection, urine volume doubled as compared to control but gradually decreased over the next 2 days (Table II). Pretreatment with Zn salts did not reduce the c-DDP-induced increase in urine volume. The urinary excretion of Pt was also maximal at one day after c-DDP injection and represented approximately 45--50°70 o f the injected Pt. An additional 5--10070 of the Pt was excreted over the next 2 days (Table II). The highest concentration of Pt measured in the urine was around 10 tag/ml. Since the activities of the enzymes, A L P , G G T and N A G were not affected by the in vitro incubation with c - D D P at concentrations up to 100 tag P t / m l urine, the concentration o f Pt excreted in the urine following c-DDP injection will not directly interfere in the measurement of these enzymes. Following c-DDP injection, only small increases in the urinary excretion of A L P , G G T and N A G were observed (Fig. 2a) and these increases were not significantly altered by Zn pretreatment (Fig. 2b). One day after c - D D P injection, the urinary excretion of creatinine was reduced by about 50070 and it remained decreased for 3 days (Table II). In the Zn-pretreated group, the c-DDP-induced decrease in creatinine excretion appeared somewhat attenuated for the first 2 days after c-DDP injection with only a 25070 decrease on day 1. However, by 3 days after c-DDP injection, creatinine excretion values were similar for both groups.
Fig. 3. Histology of rat kidney 3 days after a single injection of c-DDP (6.0 mg/kg body wt). Proximal tubules appear intact (open arrow) but tubules of the corticomedullary region (closed arrows) are necrotic with considerable loss of nuclei. H and E × 100.
120
TABLE III CONCENTRATIONS OF Pt AND MT IN RAT KIDNEY FOLLOWING c-DDP INJECTION WITH OR WITHOUT Zn PRETREATMENT All rats except controls were injected with c-DDP (6.0 mg/kg body wt) and killed 3 days later. Zn-pretreated animals were injected with ZnSO( (20 mg/kg body wt) 24 h prior to c-DDP injection. Values represent the mean ± S.D., n = 6. Treatment
Pt ~g/g)
MT 0ag/g)
Control c-DDP Zn + c-DDP
N.D. 10.38 ± 0.57 11.60 ± 2.80
32.26 ± 5.66 37.32 ± 5.18 84.04 ± 8.45 ",b
N.D., Not Determined. 'Significantly different from controls (P < 0.05). ~Significantly different from c-DDP only treated group (P < 0~5).
Pretreatment with Zn also attenuated the loss in weight which was observed three days after c-DDP injection. Whereas the body weight of rats were significantly decreased by approximately 9 0 3 days after c-DDP injection, there was no change in body weight 3 days after c-DDP injection of animals in the Zn-pretreated group (data not shown). Histological examination of the kidney after c-DDP injection revealed that in the cortex, both glomeruli and proximal tubules were intact. However, extensive damage to the tubules in the corticomedullary region was observed (Fig. 3). Despite the significant increase in renal MT concentrations following Zn pretreatment (Table liD, no attenuation of c-DDP-induced damage to the renal tubules of the corticomedullary region was observed histologically (data not shown).
GSH depletion No changes in GSH concentrations in liver were observed at any time following a single c-DDP injection (6.0 mg/kg body wt) (Table IV). In the kidney, at 24 h after c-DDP injection, renal GSH concentrations were significantly lower as compared to control (Table IV). The specific inhibitor of GSH synthesis, BSO, reduced both renal and hepatic GSH concentrations by approximately 80% within 4 h of administration and maintained significantly lower GSH concentrations in liver and kidney for 24 h (Table IV). In the BSO + c-DDP treated group, there was a small but significant decrease in the accumulation of Pt in the kidney at 24 h after c-DDP injection and at 12 and 24 h in the liver (Table IV). In contrast, MT concentrations in the kidney were significantly higher in the BSO-pretreated group compared to the non-pretreated at 12 h after c-DDP injection. By 24 h after c-DDP injection, concentra-
121
0.55 ± 0.10" 0.61 ± 0.07' 0.59 ± 0.07*
4 12 24
4 12 24
BSO
BSO + c-DDP
4.11 5.21 2.13
6.79 5.69 6.42
5.66
36.18 --. 7.01 74.68 _+ 14.48 ",b 66.78 ± 17.45 =,b
33.45 ± 29.95 ± 32.11 ±
34.85 ± 35.52 ± 35.31 ±
32.60 ±
MT (~g/g)
N.D., Not Determined. •Significantly different from Control (P < 0.05). bSignificantly different from c-DDP only group (P < 0.05).
0.60 -4" 0.08 ",b 0.49 ± 0.07 ".b 0.53 ± 0.09` .b
2.31 ± 0.40 2.26 ± 0.77 1.96 ± 0.19"
2.36 ± 0.37
GSH (~mol/g)
Kidney
4 12 24
(h)
Time
c-DDP
Control
Treatment
8.35 ± 1.17 10.51 ± 3.62 9.48 ± 1.39 b
N.D. N.D. N.D.
9.84 ± 1.16 8.03 ± 0.27 13.00 ± 0.59
N.D.
Pt (/ag/g)
1.99 ± 0.39` 4.26 ± 0.54" 5.18 ± 0.38"
2.11 + 0.41" 3.91 ± 0.77" 5.07 ± 0.62"
6.89 -+ 0.63 6.84 ± 0.63 7.67 ± 1.32
7.87 -+ 0.73
OSH ~mol/g)
Liver
10.79 ± 2.45 13.53 + 2.77 38.97 ± 9.05 ",b
9.89 ± 1.51 8.37 ± 2.63 10.31 ± 3.21
12.55 ± 2.67 11.96 -+ 2.52 19.52 ± 5.88 a
8.85 ± 1.96
MT (~g/g)
1.29 ± 0.28 b 2.13 _+ 0.35 b
N.D.
N.D. N.D. N.D.
2.61 _+ 0.34 3.36 ± 1.25
N.D.
N.D.
Pt (~g/g)
All rats except Control and BSO treatment groups were injected with c-DDP (6.0 m g / k g body wt) and killed at the indicated times. BSO-pretreated animals were injected with BSO 4 h prior to c-DDP injection. Values represent the mean ± S.D., n = 6.
CONCENTRATIONS OF GSH, MT A N D Pt IN RAT KIDNEY A N D LIVER F O L L O W I N G c-DDP I N J E C T I O N W I T H OR W I T H O U T BSO PRETREATMENT
TABLE IV
(O)
HMW
MT
LMW
160
I
120
0.6
80
0.4
40
,
0.2
\ ~..
160
°'
1 1
11
I I I |
\ %
0.6
120
li
80
~:
0,4
40
i
0.2
I0
20
FRACTION
30
40
NUMBER
Fig. 4. Cytosolicdistribution pattern of Pt (----) and Zn (. . . . . ) in kidney 24 h after c-DDP(6.0 mg/kg body wt) injection. A. No pretreatment. B. BSO-pretreated. Kidneys were homogenized in a 250 mM Sucrose-TKM buffer (pH 8.6), centrifuged at 105 000 g and a 0.3-ml aliquot of the resultant supernatant was applied on a Sephadex G-75 column. The concentrations of metals in the fractions collected from the column were analyzed by either FAAS (Pt) or AAS (Zn).
tions of MT in both kidney and liver were greater in the BSO pretreated group (Table IV). The cytosolic distribution patterns of Pt and Zn in the kidney 24 h after cDDP injection in non-pretreated and BSO pretreated rats are shown in Figs. 4a and 4b respectively. For both groups, Pt was found to be distributed primarily (,~70%) in the H M W fraction with another small peak in the LMW fraction. In contrast, changes in the protein binding profile of Zn was observed for these two groups. In the group treated with c-DDP only, Zn was associated primarily with the H M W fraction (Fig. 4a). In the c-DDP-injected group pretreated with BSO, there was a significant increase in proportion of cytosolic Zn associated with the MT fraction (Fig. 4b). There were no changes in the renal concentration o f Zn at any time after c-DDP injection in either BSO-pretreated or non-pretreated groups.
123
I~ Control C'DDP BSO + C'DDP
b
50 30 IO Control
4 hr
12 hr
24 hr
TIME (HOURS)
Fig. 5. The accumulation of PAH in rat renal slices at specific times after BSO (4 mmol/kg body wt) and/or C-DDP (6.0 mg/kg body wt) injections. Renal slices were cut from kidneys of treated animals and incubated in a medium containing PAH (7.4 X 10-~ M) for 90 rain under 100070oxygen. At the end of the incubation period, the concentrations of PAH in the slices and medium were measured and uptake was expressed as a slice to medium ratio (S:M). (Means ± S.D., n = 4) "Significantly different from control (P < 0.05) bSignificantlydifferent from c-DDP group (P < 0.05).
Up to 24 h after BSO injection, the uptake of the anion, P A H into renal slices was not significantly different f r o m control (data not shown). A small decrease in P A H uptake into renal slices o f c-DDP-injected animals occurred at 12 and 24 h after injection and at 4, 12 and 24 h after injection in BSO-pretreated animals (Fig. 5). By 24 h after c - D D P injection, P A H uptake in kidneys of BSO-pretreated animals was slightly though significantly lower as compared to the c-DDP alone injected group.
Discussion A number of studies have reported that M T synthesis is induced following the injection of c-DDP to experimental animals [8--10] and the binding of Pt from c-DDP to pre-existing MT [9,10,28]. In contrast to these studies, Mason et al. [15] failed to show any significant role of M T in the renal metabolism of Pt after c-DDP injection in rats. In a recent study [29], it was demonstrated that, although c-DDP was not a good inducer of MT, the hydrolysed form of c-DDP was able to induce M T synthesis. However, this study also demonstrated that the proportion of Pt which binds to M T was very small as compared to the binding of Pt to other intracellular proteins. The results of these studies therefore raise questions regarding the physiological relevance of the in vivo interaction between c-DDP and MT. Consequently, the role o f MT as a protective agent against the nephrotoxicity of c-DDP is unclear. Despite this, a number of reports still attribute observed reductions in c-DDP-induced nephrotoxicity to MT. In most of the studies which report the induction of MT by c-DDP, the MT was characterized only by Sephadex gel filtration which indirectly identifies the protein as the same molecular weight of M T but did not estimate MT. In the present study, M T was measured by the Ag-hem method in liver and kidney and the protein binding of Pt was investigated by Sephadex gel filtration of tissue cytosols. As measured by these methods, it appears that M T is neither induced by c-DDP injection nor 124
binds to Pt in rat kidney. Although other studies have reported the binding of Pt from c-DDP to pre-existing MT in vivo [9,10] and in vitro [28], the present study demonstrates that there is no significant Pt binding to MT in vivo even after MT induction by Zn pretreatment. These results are similar to those of Mason et al. [15], where pretreatment with Cd salts to increase intracellular MT concentrations did not result in concomitant binding of Pt to MT. Since our results suggest that there is minimal interaction between MT and the Pt from c-DDP, it was not surprising that Zn pretreatment to increase intracellular MT concentrations did not protect against the renal damage induced by cDDP. The absence of protection by MT induction against the renal toxic effects of c-DDP which we report here conflicts with a number of studies which have attributed the reduction in c-DDP-induced renal damage to the protective effects of MT. Cell lines containing high concentrations of MT were found to be resistant to the cytotoxic effects of c-DDP [11]. However, uptake of Pt into the cells was not measured and may have been one of the factors which contributed to the lower cytotoxicity of c-DDP. Indeed, the authors did conclude that in some of the c-DDP-resistant cell lines which lacked elevated MT concentrations, other factors must have contributed to the reduction of the cytotoxic effects of c-DDP. The apparent protective effects of MT against c-DDP lethality has also been studied in vivo. In studies by Naganuma et al. [12,13] protection against the lethality of c-DDP in rats was observed following pretreatment with a number of divalent metals including bismuth, cadmium, copper and zinc. However, the protection observed could not be correlated with MT concentrations in either liver or kidney. More recently, Jones and Basinger [14] showed the protective effects of the metal chelators, the dithiocarbamates, against the nephrotoxicity of c-DDP. They postulated that the dithiocarbamates may have provided protection by reducing the renal concentrations of Pt and possibly by inducing the synthesis of MT. It has been hypothesized that, in the absence of significant concentrations of MT, GSH may provide a first line of defence against the lethality of cadmium [18]. The depletion of cellular levels of this tripeptide results in a dramatic increase in toxicities of mercury (Hg) [17], Cd [18] and Cd-MT [30]. Since decreases in tissue thiol content following c-DDP treatment has been observed, it is suggested that GSH may also play a role in the binding of c-DDP [19]. In a study by Litterst et al. [10], the depletion of tissue GSH concentrations by diethylmaleate (DEM) resulted in an increased toxicity of c-DDP in rats. The enhanced toxicity was attributed to an increased binding of Pt to mitochondria and other subcellular fractions in the cell. However, it is well known that DEM itself is toxic [31] and such toxicity may have contributed to the observed increase in c-DDP-induced renal toxicity. Moreover, DEM is not a specific inhibitor of GSH synthesis and will reduce the total thiols and protein synthesis in tissues. In the present study, tissue GSH concentrations were reduced by using a less toxic but potent and specific inhibitor of GSH synthesis, BSO. Concomitant to the reduction of tissue GSH concentrations there was a slight decrease in the renal and hepatic accumulation of Pt at 24 h after c-DDP injection. A similar reduction in renal metal accumulation following GSH depletion has been observed for Hg [32] indicating that GSH may play a role in'the tissue uptake of metals. 125
A s p r e v i o u s l y m e n t i o n e d , G S H d e p l e t i o n c a n m a r k e d l y increase the toxicity o f certain metals. H o w e v e r , in the p r e s e n t s t u d y , with B S O p r e t r e a t m e n t there was o n l y a m i n o r increase in c - D D P - i n d u c e d r e n a l d a m a g e at 24 h after c - D D P inject i o n i n d i c a t i n g t h a t G S H status m a y p l a y o n l y a m i n o r role in the toxicity o f cDDP. Interestingly, at 12 a n d 24 h a f t e r c - D D P i n j e c t i o n in the k i d n e y a n d at 24 h in the liver, M T c o n c e n t r a t i o n s were significantly g r e a t e r in the B S O p r e t r e a t e d g r o u p as c o m p a r e d to the n o n - p r e t r e a t e d g r o u p ( T a b l e IV). A l t h o u g h the renal c o n c e n t r a t i o n s o f Z n were n o t c h a n g e d , t h e r e was a significant increase in the p r o p o r t i o n o f Z n a s s o c i a t e d with the M T f r a c t i o n in k i d n e y cytosol. T h e r e was no similar c h a n g e in the cytosolic d i s t r i b u t i o n o f P t which was still p r e d o m i n a n t l y b o u n d to p r o t e i n s o f H M W . T h e m e c h a n i s m s i n v o l v e d in this increase in M T c o n c e n t r a t i o n s u n d e r c o n d i t i o n s o f G S H d e p l e t i o n are n o t k n o w n . It is p o s sible t h a t the i n h i b i t i o n o f G S H synthesis after B S O t r e a t m e n t m a y result in an i n c r e a s e d a v a i l a b i l i t y o f i n t r a c e l l u l a r cysteine for M T synthesis. A c h a n g e in the d e g r a d a t i o n rate o f M T to m a i n t a i n the tissue t h i o l levels also c a n n o t be ruled o u t u n d e r these e x p e r i m e n t a l c o n d i t i o n s . F u r t h e r studies are r e q u i r e d to d e l i n e a t e the m e c h a n i s m s i n v o l v e d a n d the significance o f G S H in the m e t a b o l i s m o f M T in the kidney.
Acknowledgements T h e a u t h o r s wish t o t h a n k M r . R o n N o s e w o r t h y for s t a n d a r d i z i n g the F A A S m e t h o d o f analysis for P t . This w o r k was s u p p o r t e d b y g r a n t s f r o m the M e d i c a l Research Council of Canada.
References 1
M. Dentino, F. Luft, M.N. Yum, S.D. Williams and L.H. Einhorn, Long term effect of cisdiamminedichloride platinum (CDDP) on renal function and structure in man. Cancer., 41 (1978) 1274. 2 R.S. Goldstein and G.H. Mayor, The nephrotoxicity of cisplatin. Life Sci., 32 (1983) 685. 3 M.W. Weiner and C. Jacobs, Mechanism of cisplatin nephrotoxicity. Fed. Proc., 42 (1983) 2974. 4 D.D. Choie, D.S. Longnecker and A.A. Del Campo, Acute and chronic cisplatin nephropathy in rats. Lab. Invest., 44 (1981) 397. 5 G.F. Nordberg, Effects of acute and chronic cadmium exposure on the testicles of mice with special reference to protective effects of metallothionein. Environ. Physiol., 1 (1971) 171. 6 M. Webb, Binding of cadmium ions by rat liver and kidney. Biochem. Pharmacol., 21 (1972) 2751. 7 M.G. Cherian, The synthesis of metallothionein and cellular adaptation to metal toxicity in primary rat kidney epithelial cell cultures. Toxicology, 17 (1980) 225. 8 R.P. Sharma and L.R. Edwards, Cis-platinum: Subcellular distribution and binding to cytosolic ligands. Biochem. Pharmacol., 32 (1983) 2665. 9 A.J. Zelazowski, J.S. Garvey and J.D. Hoeschele, In vivo and in vitro binding of platinum to metaliothionein. Arch. Biochem. Biophys., 229 (1984)246. 10 C.L. Litterst, F. Bertolero and J. Uozumi, The role of glutathione and metallothionein in the toxicity and subcellular binding of cisplatin, in D.C.H. McBrien and T.F. Slater (Eds.), Biochemical Mechanisms of Platinum Antitumour Drugs, IRL Press, Oxford, 1986, p. 227.
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A. Bakka, L. Endresen, B.B.S. Johnsen, P.D. Edminson and H.E. Rugstad, Resistance against cis-dichiorodiammineplatinum in cultured cells with a high content of metallothionein. Toxicol. Appl. Pharmacol., 61 (1981) 215. A. Naganuma, M. Satoh and N. lmura, Prevention of lethal and renal toxicity of c/s-diamminedichloroplatinum (II) by induction of metaUothionein synthesis without compromising its antitumour activity in mice. Cancer Res., 47 0987) 983. A. Naganuma, M. Satoh, Y. Koyama and N. lmura, Protective effect of metaliothionein inducing metals on lethal toxicity of c/s-diamminedichloroplatinum in mice. Toxicol. Lett., 24 (1985) 203. M.M. Jones and M.A. Basinger, Control of nephrotoxicity in the rat during repeated c/s-platinum treatments. J. Appl. Toxicol., 9 (1989) 229. R. Mason, I.R. Edwards and S.J. McLaren, Interaction of platinum with metallothionein-like ligands in the rat kidney after administration of c/s-dichlorodiammine platinum ll. Chem-Biol. Interact., 49 (1984) 165. C.E. Hildebrand, J.K. Griffith, R.A. Tobey, R.A Waiters and M.D. Enger, Molecular mechanisms of cadmium detoxification in cadmium-resistant cultured cells: Role of metallothionein and other inducible factors, in E.C. Foulkes (Ed.), Biological Roles of Metallothionein, Elsevier/North Holland, Amsterdam, 1982, p. 279. J. Baggett and W.O. Berndt, The effect of depletion of non-protein sulfhydryls by diethyl maleate plus buthionine sulfoxime on renal uptake of mercury in the rat. Toxicol. Appl. Pharmacol., 83 0986) 556. R.R. Singhal, M.E. Anderson and A. Meister, Glutathione, a first line of defense against cadmium toxicity. FASEB J., 1 (1987) 220. J. Levi, C. Jacobs, S.M. Kalman, M. McTigue and M.W. Weiner, Mechanism of c/s-platinum nephrotoxicity: I Effects of sulfhydryl groups in rat kidneys. J. Pharmacol. Expel Therap., 213 (1981) 545. D.C. Dobyan, J. Levi, C. Jacobs, J. Kosek and M.W. Weiner, Mechanisms of c/s-platinum nephrotoxicity: II Morphologic observations. J. Pharmacol. Expel Ther., 213 (1980) 551. A.M. Scheuhammer and M.G. Cherian, Quantification of metallothioneins by a silver-saturation method. Toxicol. Appl. Pharmacol., 82 (1986) 417. W.E. Stroo and J.B. Hook, Enzymes of renal origin in urine as indicators of nephrotoxicity. Toxicol. Appl. Pharmacol., 39 (1977) 423. R.G. Price, Urinary enzymes, nephrotoxicity and renal disease. Toxicology, 23 (1982) 99. M.A. Smith, J.H. Smith, C.L. Litterst, M.P. Copley, J. Uozumi and M.R. Boyd, In vivo biochemical indices of nephrotoxicity of platinum analogs, tetraplatin, CHIP and cisplatin in the Fischer 344 rat. Fund. Appl. Toxicol., 10 (1988) 62. C.A.M. Suzuki and M.G. Cherian, Renal toxicity of cadmium-metallothionein and enzymuria in rats. J. Pharmacol. Exp. Ther., 240 0988) 314. D.J. Reed and M.W. Fariss, Glutathione depletion and susceptibility. Pharmacol. Rev., 36 (1984) 255. C.A.M. Suzuki and M.G. Cherian, Effects of cadmium-metallothionein on renal organic ion transport and lipid peroxidation in rats. J. Biochem. Toxicol., 3 (1988) l 1. J. Bongers, J.H. Bell and D.E. Richardson, Platinum(II) binding to metallothioneins. J. lnorg. Biochem., 34 (1988) 55. P.G. Farnworth, B.L. Hillcoat, I.A.G. Roos, Metallothionein induction in mouse tissues by c/sdiamminedichloroplatinum (II) and its hydrolysis products. Chem-Biol. Interact., 69 (1989) 319. C.A.M. Suzuki and M.G. Cherian, Renal glutathione depletion and nephrotoxicity of cadmiummetallothionein in rats. Toxicol. Appl. Pharmacol., 98 (1989) 544. M.E. Davis, W.O. Berndt and H.M. Mehendale, Effects of cysteine and diethylmaleate pretreatmerits on renal function and response to a nephrotoxicant. Arch. Toxicol., 59 (1986) 7. W.O. Berndt, J. Baggett, A. Blacker and M. Houser, Renal glutathione and mercury uptake by kidney. Fundam. Appl. Toxicol., 5 (1985) 832.
127
The interactions of cis-diamminedichloroplatinum with metallothionein and glutathione in rat liver and kidney Catherine A.M. Suzuki* and M.G. Cherian** Departments of Pharmacology and Toxicology and Pathology, University of Western Ontario, London, Ontario N6A 5C1 (Canada) (Received January 23rd, 1990; accepted May 1lth, 1990)
Summary The involvement of metallothionein (MT) in the nephrotoxicity of cis-diamminedichloroplatinum (c-DDP) was investigated in rats using enzyme excretion and histology as indicators of renal damage. In addition, the effects of renal glutathione (GSH) depletion on the nephrotoxicity of c-DDP was assessed by organic anion transport in renal cortical slices. A dose of 6.0 mg c-DDP/kg body wt, i.p. was administered to rats either as a single injection of 6.0 mg/kg or as six daily injections of 1.0 mg/kg. Concentrations of platinum (Pt) after c-DDP injection in both dosing regimens were approximately 12 i~g/g in kidney and 2 pg/g in liver. However, there were no increases in either hepatic or renal concentrations of MT after both series of c-DDP injections. Fractionation of kidney cytosols from c-DDP injected rats on Sephadex G-75 columns revealed that 60--70070 of cytosolic Pt was associated with proteins of high molecular weight and 15--20070 of the Pt associated with the low molecular weight ligands. No discernable Pt peak was detected in the elution volume of MT. Pretreatment of rats with ZnSO~ increased both hepatic and renal concentrations of MT, but there was no Pt associated with the MT fraction after a subsequent injection of cDDP. Small increases in the urinary excretion of the lysosomal enzyme, N-acetyl-p-D-glucosaminidase and two brush border enzymes, alkaline phosphatase and y-glutamyltranspeptidase were observed 2 and 3 days after a single injection of c-DDP (6.0 mg/kg body wt, i.p.). Urinary creatinine excretion decreased by 50°70 1 day after c-DDP injection and continued to decrease for the next 2 days. On the third day after c-DDP treatment, a small but significant decrease in body weight was also observed in the c-DDP injected animals. Pretreatment with Zn did not alter the c-DDP-induced enzymuria or renal tubular damage but slightly attenuated both the decrease in creatinine excretion and the loss in body weight. Uptake of the organic anion, p-aminohippuric acid (PAH) was reduced at 12 and 24 h after cDDP injection. Reduction of tissue GSH concentrations by pretreatment with buthionine sulfoxime (BSO), resulted in only a slight increase in the c-DDP-induced inhibition of PAH uptake at 24 h after c-DDP injection.
*Present Address: Drug Toxicology Division, Health Protection Branch, Sir F.G. Banting Research Centre, Tunney's Pasture, Ottawa, Ontario, Canada. K1A OL2 **To whom all correspondence should be addressed. 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
113
These results suggest that, in rats, neither MT nor GSH appear to play major roles in the binding or nephrotoxicityof c-DDP. Key words: C/s-diamminedichloroplatinum;Metallothionein; Glutathione; Nephrotoxicity Introduction
Cis-diamminedichloroplatinum (c-DDP) is a member of a group of antineoplastic agents containing the divalent metal, platinum (Pt). Although the use of cDDP often results in the development of deleterious side effects, the use of this potent chemotherapeutic agent is primarily limited by a dose-related nephrotoxicity [1--3]. Since the renal damage caused by c-DDP treatment is similar to some of the toxic effects of metals, it has been hypothesized that the renal damage induced by c-DDP may be mediated by the Pt moiety itself [4]. As a result, it has been suggested that the high cysteine-containing intracellular protein, metallothionein (MT) which is known to play an important role in the detoxification of cadmium (Cd) [5--7], may also reduce the nephrotoxicity of c-DDP. The induction of MT synthesis and binding of Pt to MT following c-DDP injection into experimental animals has been reported by a number of investigators [8--10] and consequently the induction of MT as a protective mechanism against the nephrotoxicity of c-DDP has been investigated [11--14]. However, in a study by Mason et al. [15], following c-DDP injection to rats, there was no induction of MT synthesis or any binding of the Pt with pre-existing MT. Thus, the involvement of MT in the metabolism and the renal toxicity of c-DDP remains unclear and was further investigated in the present study. Glutathione (GSH), the major non-protein thiol source in the cell, plays an important role in the metabolism and detoxifieation of a number of chemicals and its potential role in metal detoxification has been recently investigated [16-18]. It has been hypothesized that the high levels of GSH in kidney and liver may provide a readily accessible pool of thiol binding sites for metal binding prior to the synthesis of MT. Since GSH has been implicated in the metabolism of c-DDP [19], the effects of tissue GSH depletion on the toxicity of c-DDP was studied. Methods
M T induction and Pt binding Male Sprague--Dawley rats (200--250 g) (Charles River, Canada) used in this study were maintained on a 12 h light and dark cycle, fed standard Purina rat chow and given water ad libitum. All the c-DDP (Bristol Myers, Syracuse, N.Y.) solutions were freshly prepared just prior to injection. A dose of 6.0 mg c - D D P / kg body wt, i.p. was chosen for the present study since a similar dose was used in a previous report to demonstrate MT induction [8]. However, since this dose has also been shown to result in pathological changes to the kidney [20], the same total dose was injected into rats as 6 injections of 1.0 mg c-DDP/kg body wt, i.p. over 6 days in an attempt to reduce the immediate toxic effects of c-DDP.
114
Animals were killed under pentobarbital anaesthesia by exsanguination from the abdominal aorta at 24 h after the last injection of c-DDP and livers and kidneys were removed and analyzed for Pt content by flameless atomic absorption spectrometry (FAAS). Tissue samples (approx. 0.5 g) were digested for 24 h in nitric acid (15 N) and the resultant solution was evaporated to near dryness by gentle heating. The residue was resuspended in 1 N HC1, again evaporated by heating, the residue resuspended in 0.1 N HCI and again evaporated to near dryness. The sample was diluted to a final volume of 5.0 ml with 0.1 N HCI and Pt concentrations in the samples were measured by FAAS on a Varian Spectra 30 (Varian Canada, Georgetown, Ontario). The parameters for Pt analysis by FAAS were as follows: Lamp current, 7.0 mAmps; slit width, 0.2 nm; wavelength, 265.9 nm; background correction on. Tissue MT concentrations were determined using the silver saturation method [21]. To examine the protein binding profile of Pt in the cytosol, immediately after the animal was killed, the kidney was removed and a 0.5 g sample of the kidney was homogenized in 4 vols. of a 250 mM sucrose-Tris (50 mM)-KC1 (25 mM)MgCI 2 (5 raM) (TKM) buffer, pH 8.6 and the homogenate was centrifuged at 105 000 g for 1 h. An aliquot of the resultant supernatant (0.3 ml) was fractionated on a standardized Sephadex G-75 column (0.9 cm x 60.0 cm) and eluted with 10 mM TrismHCl buffer (pH 8.6). The fractions (1.0 ml) were analyzed for Pt by FAAS. To examine the in vivo binding of Pt to MT, rats were pretreated with ZnSO 4 (20 mg Zn/kg body wt, s.c.) 24 h prior to a single c-DDP injection (6 mg/kg body wt, i.p.). Twenty-four hours after c-DDP injection, animals were killed by exsanguination and tissues (liver and kidney) were analyzed for Pt, Zn and MT. Kidney cytosols were fractionated on standardized Sephadex G-75 columns and fractions were analyzed for Pt and Zn as previously described.
Urinary enzyme excretion The measurement of urinary enzymes has been used as a sensitive, non-invasive method for assessing renal damage of a number of nephrotoxicants [22,23] and it has been suggested that urinary enzymes may provide a sensitive indicator of c-DDP-induced renal damage [24]. Since injected c-DDP is excreted primarily in urine, it is important to determine the effects of c-DDP on enzyme activity in urine. The direct in vitro effect of c-DDP on the activities of the enzymes, alkaline phosphatase (ALP), r-glutamyltranspeptidase (GGT) and N-acetyl-/3-D-glucosaminidase (NAG) in urine was examined by incubating 2-ml aliquots of urine for 2 h at 4°C with and without addition of c-DDP in concentrations of 10--100 /~g Pt/ml of urine. Male Sprague--Dawley rats (250--270 g) were housed individually in plastic Nalgene metabolic cages. For 3 days prior to treatment, 24 h urine samples were collected over ice and analyzed for creatinine, ALP, GGT and NAG as previously described [25]. These values represented control urinary enzyme excretion levels. After a single injection of c-DDP (6.0 mg/kg body wt, i.p.), 24-h urine samples were collected for another 3 days and urinary enzyme levels and Pt were 115
measured. Enzyme excretion values were calculated as the amount excreted per 24-h period and expressed as a percentage of control. Rats were killed at the end of 3 days and body weight and the renal concentrations of Pt, Zn and MT were measured. A section of kidney was fixed in neutral 10% formalin, embedded in paraffin, and stained with haematoxylin and eosin (H&E) for examination by light microscopy. A second group of rats were injected with ZnSO 4 (20 mg Zn/kg body wt, s.c.) 24 h before the single c-DDP injection (6.0 mg/kg body wt, i.p.) and urinary enzyme excretion, kidney morphology and tissue Pt, Zn and MT concentrations were analyzed as described before. GSH Depletion Tissue GSH concentrations were decreased in male Sprague--Dawley rats (200 --250 g), by treatment with a specific inhibitor of GSH biosynthesis, buthionine sulfoxime (BSO) (Sigma Chemical Co., St. Louis, MO.) (4 mmol/kg body wt, s.c.). Four hours after BSO injection, rats were injected with c-DDP (a single injection of 6.0 mg/kg body wt, i.p.) and killed by decapitation 4, 12 or 24 h later. Kidney and liver concentrations of Pt, GSH and MT were measured. Rats were killed at the same time of day since tissue GSH content is subject to diurnal variations [26]. Damage to the kidney was assessed by measuring the uptake of paminohippuric acid (PAH) in renal slices as previously described [27l. Kidney samples were homogenized and cytosols were prepared for fractionation on a Sephadex G-75 column and column fractions were analyzed for Pt and Zn as described before. Statistical analysis Statistical differences were calculated with Student's t-test or one-way analysis of variance (ANOVA) where required. Multiple comparison testing was performed using the Student-Neuman-Keuls test. Differences were considered statistically significant if they achieved a significance level of P < 0.05. Results
M T induction and Pt binding After the injection of rats with c-DDP (6.0 mg/kg) by either dosing regimen, Pt concentrations in tissues were approximately 10--13 /ag/g in kidney and 2--3 /ag/g in liver (Table I). The concentrations of Zn and MT in these tissues were similar to controls (Table I). Kidney cytosols from individual rats injected with c-DDP were fractionated on a Sephadex G-75 column. A typical elution profile is shown in Fig. 1 for Pt in a kidney cytosol of a rat which had been injected with c-DDP at a dose of 1.0 mg/ kg each day for 6 days. Approximately 60--70% of cytosolic Pt was associated with proteins of high molecular weight (HMW) with 15--20% associated with the
116
TABLE 1 RENAL AND HEPATIC CONCENTRATIONS OF Pt, Zn AND MT IN RATS AFTER c-DDP INJECTION WITH OR WITHOUT Zn PRETREATMENT All animals except controls were injected with c-DDP and killed 24 h after the last injection. The numbers in parentheses represent the number of injections and the dose of c-DDP of each injection. Zn-pretreated rats were injected with ZnSO, (20 mg Zn/kg body wt.) 24 h prior to c-DDP injection. Values represent the mean ± S.D., n = 6. Treatment
Pt (/ag/g tissue)
Zn ~g/g tissue)
MT (~g/g tissue)
Control
Kidney Liver
N.D. N.D.
26.79 ± 5.24 30.54 ± 2.68
32.60 ± 8.85 ±
5.66 1.96
c-DDP (6x 1 mg/kg)
Kidney
10.02 ± 2.31
33.79 ± 6.07
29.27 ±
9.41
2.45 ± 0.09
31.65 ± 2.49
7.74 -4- 1.36
13.00 ± 0.77
26.63 ± 3.97
35.32 _+ 6.41
3.36 ± 0.35
33.92 ± 3.73
15.52 ±
10.81 _+ 1.52
28.99 ± 1.65
86.55 ± 17.82"
2.91 ± 0.44
41.07 ± 4.71"
150.77 _+ 15.41"
Liver
c-DDP 0×6 mg/kg)
Kidney
Zn + c-DDP (6x 1 mg/kg)
Kidney
Liver
Liver
5.22
N.D. Not determined.
*Significantly different from control (P < 0.05).
low m o l e c u l a r weight ( L M W ) ligands. T h e r e m a i n d e r o f the P t was d i s t r i b u t e d b e t w e e n the H M W a n d L M W regions with n o a p p a r e n t p e a k in the region where M T is eluted (10,000 d a l t o n s ) . T h e cytosolic d i s t r i b u t i o n o f P t a f t e r the single high d o s e (6.0 m g / k g ) i n j e c t i o n o f c - D D P was similar ( d a t a n o t shown). Recoveries o f P t in the f r a c t i o n s were b e t w e e n 92--100%0 o f the t o t a l a m o u n t o f P t a p p l i e d to the c o l u m n s . P r e t r e a t m e n t with Z n at 24 h p r i o r to c - D D P i n j e c t i o n resulted in significant increases in b o t h renal a n d h e p a t i c M T c o n c e n t r a t i o n s as c o m p a r e d to the c - D D P a l o n e injected g r o u p ( T a b l e I). T h e a c c u m u l a t i o n o f P t in liver a n d k i d n e y was n o t altered b y Z n p r e t r e a t m e n t . T h e gel f i l t r a t i o n o f cytosols f r o m kidneys o f a c - D D P injected rat p r e t r e a t e d with Z n r e v e a l e d t h a t a l t h o u g h a p p r o x i m a t e l y 15% o f cytosolic Z n was e l u t e d in the r e g i o n o f M T , there was no m e a s u r a b l e a m o u n t o f P t a s s o c i a t e d with this p e a k ( d a t a n o t s h o w n ) . S i m i l a r to the results in Fig. 1, the m a j o r i t y o f t h e P t ( 6 0 % ) was f o u n d with the H M W p r o t e i n s with a n o t h e r 2 2 % o f the P t a s s o c i a t e d with the L M W l i g a n d s a n d the r e m a i n i n g P t d i s t r i b u t e d f a i r l y u n i f o r m l y b e t w e e n the H M W a n d L M W f r a c t i o n s ( d a t a not shown).
117
oo
17.5 40.0 28.5 28.0
-+ +. +. ---
3.8 11.8 a 4.21 13.7
N.D. 433.5 --4-- 55.9 34.0 _+ 3.7 19.2 +. 4.1
10.9 5.6 3.3 4.9
-+ -+ +. +.
0.8 1.6 "b 0.8 a'b 1.9~
15.8 41.2 25.8 25.3
+- 2.9 +. 12.5 ° -+ 8.9 ~ +. 6.9 ~
Volume (mi/day)
Creatinine (mg/day)
Volume (ml/day)
Pt (~g/day)
Zn + c-DDP
c-DDP
Treatment
N.D. Not Determined. •Significantly different from control (P < 0.05). bSignificantly different from Zn + c-DDP group.
Control 1 2 3
Day After c-DDP Injection
N.D. 480.5 +. 39.5 42.7 +. 9.0 23.8 +. 3.5
Pt Og/day)
9.7 7.5 5.9 4.5
+++. +.
1.2 1.6 1.3 I 0.8 a
Creatinine (rag/day)
Rats were injected with c-DDP (6.0 m g / k g body wt.) and killed 3 days later. Zn-pretreated animals were injected with ZnSO 4 (20 mg Z n / k g body wt.) 24 h prior to c-DDP injection. Controls are values before c-DDP injection. Values represent the mean +. S.D., n = 6.
DAILY URINE VOLUMES AND U R I N A R Y E X C R E T I O N OF Pt AND CREATININE F O L L O W I N G c-DDP INJECTION
TABLE II
\
~ ,o l
~
so 2o
I 20
IO
30
40
FRACTION NUMBER Fig. 1. The cytosolic distribution patterns of Pt in kidney after the injection of c-DDP (1 mg/kg body wt) daily for 6 days. Twenty-four hours after the last injection, rats were sacrificed, kidneys removed and homogenized in a 250 mM sucrose-TKM buffer (pH 8.6), centrifuged at 105 000 g and an aliquot of the resultant supernatant applied on a Sephadex G-75 column. Fractions were analyzed for Pt by FAAS. 300
A
I
.
I
. GGT
=
NAG
COOP
0
I
2
3
4
!4
/
g ZnSO 4
| i f COOP
I 2
I 3
I 4
TIME (Doys) Fig. 2. Urinary excretion profiles of GGT (A), ALP ( , ) and NAG (O) after a single injection of cDDP (6.0 mg/kg body wt). (A) Saline pretreated. (B) ZnSO4 (20 mg Zn/kg body wt) pretreated 24 h prior to c-DDP injection. Twenty-four hour urine samples were collected over ice and analyzed for ALP, GGT and NAG activities. Activities of the enzymes were expressed as percentages of control values. (Means ± S.D., n = 6) *Significantly different from control (P < 0.05).
119
Urinary enzyme excretion Within the first 24-h period after c-DDP injection, urine volume doubled as compared to control but gradually decreased over the next 2 days (Table II). Pretreatment with Zn salts did not reduce the c-DDP-induced increase in urine volume. The urinary excretion of Pt was also maximal at one day after c-DDP injection and represented approximately 45--50°70 o f the injected Pt. An additional 5--10070 of the Pt was excreted over the next 2 days (Table II). The highest concentration of Pt measured in the urine was around 10 tag/ml. Since the activities of the enzymes, A L P , G G T and N A G were not affected by the in vitro incubation with c - D D P at concentrations up to 100 tag P t / m l urine, the concentration o f Pt excreted in the urine following c-DDP injection will not directly interfere in the measurement of these enzymes. Following c-DDP injection, only small increases in the urinary excretion of A L P , G G T and N A G were observed (Fig. 2a) and these increases were not significantly altered by Zn pretreatment (Fig. 2b). One day after c - D D P injection, the urinary excretion of creatinine was reduced by about 50070 and it remained decreased for 3 days (Table II). In the Zn-pretreated group, the c-DDP-induced decrease in creatinine excretion appeared somewhat attenuated for the first 2 days after c-DDP injection with only a 25070 decrease on day 1. However, by 3 days after c-DDP injection, creatinine excretion values were similar for both groups.
Fig. 3. Histology of rat kidney 3 days after a single injection of c-DDP (6.0 mg/kg body wt). Proximal tubules appear intact (open arrow) but tubules of the corticomedullary region (closed arrows) are necrotic with considerable loss of nuclei. H and E × 100.
120
TABLE III CONCENTRATIONS OF Pt AND MT IN RAT KIDNEY FOLLOWING c-DDP INJECTION WITH OR WITHOUT Zn PRETREATMENT All rats except controls were injected with c-DDP (6.0 mg/kg body wt) and killed 3 days later. Zn-pretreated animals were injected with ZnSO( (20 mg/kg body wt) 24 h prior to c-DDP injection. Values represent the mean ± S.D., n = 6. Treatment
Pt ~g/g)
MT 0ag/g)
Control c-DDP Zn + c-DDP
N.D. 10.38 ± 0.57 11.60 ± 2.80
32.26 ± 5.66 37.32 ± 5.18 84.04 ± 8.45 ",b
N.D., Not Determined. 'Significantly different from controls (P < 0.05). ~Significantly different from c-DDP only treated group (P < 0~5).
Pretreatment with Zn also attenuated the loss in weight which was observed three days after c-DDP injection. Whereas the body weight of rats were significantly decreased by approximately 9 0 3 days after c-DDP injection, there was no change in body weight 3 days after c-DDP injection of animals in the Zn-pretreated group (data not shown). Histological examination of the kidney after c-DDP injection revealed that in the cortex, both glomeruli and proximal tubules were intact. However, extensive damage to the tubules in the corticomedullary region was observed (Fig. 3). Despite the significant increase in renal MT concentrations following Zn pretreatment (Table liD, no attenuation of c-DDP-induced damage to the renal tubules of the corticomedullary region was observed histologically (data not shown).
GSH depletion No changes in GSH concentrations in liver were observed at any time following a single c-DDP injection (6.0 mg/kg body wt) (Table IV). In the kidney, at 24 h after c-DDP injection, renal GSH concentrations were significantly lower as compared to control (Table IV). The specific inhibitor of GSH synthesis, BSO, reduced both renal and hepatic GSH concentrations by approximately 80% within 4 h of administration and maintained significantly lower GSH concentrations in liver and kidney for 24 h (Table IV). In the BSO + c-DDP treated group, there was a small but significant decrease in the accumulation of Pt in the kidney at 24 h after c-DDP injection and at 12 and 24 h in the liver (Table IV). In contrast, MT concentrations in the kidney were significantly higher in the BSO-pretreated group compared to the non-pretreated at 12 h after c-DDP injection. By 24 h after c-DDP injection, concentra-
121
0.55 ± 0.10" 0.61 ± 0.07' 0.59 ± 0.07*
4 12 24
4 12 24
BSO
BSO + c-DDP
4.11 5.21 2.13
6.79 5.69 6.42
5.66
36.18 --. 7.01 74.68 _+ 14.48 ",b 66.78 ± 17.45 =,b
33.45 ± 29.95 ± 32.11 ±
34.85 ± 35.52 ± 35.31 ±
32.60 ±
MT (~g/g)
N.D., Not Determined. •Significantly different from Control (P < 0.05). bSignificantly different from c-DDP only group (P < 0.05).
0.60 -4" 0.08 ",b 0.49 ± 0.07 ".b 0.53 ± 0.09` .b
2.31 ± 0.40 2.26 ± 0.77 1.96 ± 0.19"
2.36 ± 0.37
GSH (~mol/g)
Kidney
4 12 24
(h)
Time
c-DDP
Control
Treatment
8.35 ± 1.17 10.51 ± 3.62 9.48 ± 1.39 b
N.D. N.D. N.D.
9.84 ± 1.16 8.03 ± 0.27 13.00 ± 0.59
N.D.
Pt (/ag/g)
1.99 ± 0.39` 4.26 ± 0.54" 5.18 ± 0.38"
2.11 + 0.41" 3.91 ± 0.77" 5.07 ± 0.62"
6.89 -+ 0.63 6.84 ± 0.63 7.67 ± 1.32
7.87 -+ 0.73
OSH ~mol/g)
Liver
10.79 ± 2.45 13.53 + 2.77 38.97 ± 9.05 ",b
9.89 ± 1.51 8.37 ± 2.63 10.31 ± 3.21
12.55 ± 2.67 11.96 -+ 2.52 19.52 ± 5.88 a
8.85 ± 1.96
MT (~g/g)
1.29 ± 0.28 b 2.13 _+ 0.35 b
N.D.
N.D. N.D. N.D.
2.61 _+ 0.34 3.36 ± 1.25
N.D.
N.D.
Pt (~g/g)
All rats except Control and BSO treatment groups were injected with c-DDP (6.0 m g / k g body wt) and killed at the indicated times. BSO-pretreated animals were injected with BSO 4 h prior to c-DDP injection. Values represent the mean ± S.D., n = 6.
CONCENTRATIONS OF GSH, MT A N D Pt IN RAT KIDNEY A N D LIVER F O L L O W I N G c-DDP I N J E C T I O N W I T H OR W I T H O U T BSO PRETREATMENT
TABLE IV
(O)
HMW
MT
LMW
160
I
120
0.6
80
0.4
40
,
0.2
\ ~..
160
°'
1 1
11
I I I |
\ %
0.6
120
li
80
~:
0,4
40
i
0.2
I0
20
FRACTION
30
40
NUMBER
Fig. 4. Cytosolicdistribution pattern of Pt (----) and Zn (. . . . . ) in kidney 24 h after c-DDP(6.0 mg/kg body wt) injection. A. No pretreatment. B. BSO-pretreated. Kidneys were homogenized in a 250 mM Sucrose-TKM buffer (pH 8.6), centrifuged at 105 000 g and a 0.3-ml aliquot of the resultant supernatant was applied on a Sephadex G-75 column. The concentrations of metals in the fractions collected from the column were analyzed by either FAAS (Pt) or AAS (Zn).
tions of MT in both kidney and liver were greater in the BSO pretreated group (Table IV). The cytosolic distribution patterns of Pt and Zn in the kidney 24 h after cDDP injection in non-pretreated and BSO pretreated rats are shown in Figs. 4a and 4b respectively. For both groups, Pt was found to be distributed primarily (,~70%) in the H M W fraction with another small peak in the LMW fraction. In contrast, changes in the protein binding profile of Zn was observed for these two groups. In the group treated with c-DDP only, Zn was associated primarily with the H M W fraction (Fig. 4a). In the c-DDP-injected group pretreated with BSO, there was a significant increase in proportion of cytosolic Zn associated with the MT fraction (Fig. 4b). There were no changes in the renal concentration o f Zn at any time after c-DDP injection in either BSO-pretreated or non-pretreated groups.
123
I~ Control C'DDP BSO + C'DDP
b
50 30 IO Control
4 hr
12 hr
24 hr
TIME (HOURS)
Fig. 5. The accumulation of PAH in rat renal slices at specific times after BSO (4 mmol/kg body wt) and/or C-DDP (6.0 mg/kg body wt) injections. Renal slices were cut from kidneys of treated animals and incubated in a medium containing PAH (7.4 X 10-~ M) for 90 rain under 100070oxygen. At the end of the incubation period, the concentrations of PAH in the slices and medium were measured and uptake was expressed as a slice to medium ratio (S:M). (Means ± S.D., n = 4) "Significantly different from control (P < 0.05) bSignificantlydifferent from c-DDP group (P < 0.05).
Up to 24 h after BSO injection, the uptake of the anion, P A H into renal slices was not significantly different f r o m control (data not shown). A small decrease in P A H uptake into renal slices o f c-DDP-injected animals occurred at 12 and 24 h after injection and at 4, 12 and 24 h after injection in BSO-pretreated animals (Fig. 5). By 24 h after c - D D P injection, P A H uptake in kidneys of BSO-pretreated animals was slightly though significantly lower as compared to the c-DDP alone injected group.
Discussion A number of studies have reported that M T synthesis is induced following the injection of c-DDP to experimental animals [8--10] and the binding of Pt from c-DDP to pre-existing MT [9,10,28]. In contrast to these studies, Mason et al. [15] failed to show any significant role of M T in the renal metabolism of Pt after c-DDP injection in rats. In a recent study [29], it was demonstrated that, although c-DDP was not a good inducer of MT, the hydrolysed form of c-DDP was able to induce M T synthesis. However, this study also demonstrated that the proportion of Pt which binds to M T was very small as compared to the binding of Pt to other intracellular proteins. The results of these studies therefore raise questions regarding the physiological relevance of the in vivo interaction between c-DDP and MT. Consequently, the role o f MT as a protective agent against the nephrotoxicity of c-DDP is unclear. Despite this, a number of reports still attribute observed reductions in c-DDP-induced nephrotoxicity to MT. In most of the studies which report the induction of MT by c-DDP, the MT was characterized only by Sephadex gel filtration which indirectly identifies the protein as the same molecular weight of M T but did not estimate MT. In the present study, M T was measured by the Ag-hem method in liver and kidney and the protein binding of Pt was investigated by Sephadex gel filtration of tissue cytosols. As measured by these methods, it appears that M T is neither induced by c-DDP injection nor 124
binds to Pt in rat kidney. Although other studies have reported the binding of Pt from c-DDP to pre-existing MT in vivo [9,10] and in vitro [28], the present study demonstrates that there is no significant Pt binding to MT in vivo even after MT induction by Zn pretreatment. These results are similar to those of Mason et al. [15], where pretreatment with Cd salts to increase intracellular MT concentrations did not result in concomitant binding of Pt to MT. Since our results suggest that there is minimal interaction between MT and the Pt from c-DDP, it was not surprising that Zn pretreatment to increase intracellular MT concentrations did not protect against the renal damage induced by cDDP. The absence of protection by MT induction against the renal toxic effects of c-DDP which we report here conflicts with a number of studies which have attributed the reduction in c-DDP-induced renal damage to the protective effects of MT. Cell lines containing high concentrations of MT were found to be resistant to the cytotoxic effects of c-DDP [11]. However, uptake of Pt into the cells was not measured and may have been one of the factors which contributed to the lower cytotoxicity of c-DDP. Indeed, the authors did conclude that in some of the c-DDP-resistant cell lines which lacked elevated MT concentrations, other factors must have contributed to the reduction of the cytotoxic effects of c-DDP. The apparent protective effects of MT against c-DDP lethality has also been studied in vivo. In studies by Naganuma et al. [12,13] protection against the lethality of c-DDP in rats was observed following pretreatment with a number of divalent metals including bismuth, cadmium, copper and zinc. However, the protection observed could not be correlated with MT concentrations in either liver or kidney. More recently, Jones and Basinger [14] showed the protective effects of the metal chelators, the dithiocarbamates, against the nephrotoxicity of c-DDP. They postulated that the dithiocarbamates may have provided protection by reducing the renal concentrations of Pt and possibly by inducing the synthesis of MT. It has been hypothesized that, in the absence of significant concentrations of MT, GSH may provide a first line of defence against the lethality of cadmium [18]. The depletion of cellular levels of this tripeptide results in a dramatic increase in toxicities of mercury (Hg) [17], Cd [18] and Cd-MT [30]. Since decreases in tissue thiol content following c-DDP treatment has been observed, it is suggested that GSH may also play a role in the binding of c-DDP [19]. In a study by Litterst et al. [10], the depletion of tissue GSH concentrations by diethylmaleate (DEM) resulted in an increased toxicity of c-DDP in rats. The enhanced toxicity was attributed to an increased binding of Pt to mitochondria and other subcellular fractions in the cell. However, it is well known that DEM itself is toxic [31] and such toxicity may have contributed to the observed increase in c-DDP-induced renal toxicity. Moreover, DEM is not a specific inhibitor of GSH synthesis and will reduce the total thiols and protein synthesis in tissues. In the present study, tissue GSH concentrations were reduced by using a less toxic but potent and specific inhibitor of GSH synthesis, BSO. Concomitant to the reduction of tissue GSH concentrations there was a slight decrease in the renal and hepatic accumulation of Pt at 24 h after c-DDP injection. A similar reduction in renal metal accumulation following GSH depletion has been observed for Hg [32] indicating that GSH may play a role in'the tissue uptake of metals. 125
A s p r e v i o u s l y m e n t i o n e d , G S H d e p l e t i o n c a n m a r k e d l y increase the toxicity o f certain metals. H o w e v e r , in the p r e s e n t s t u d y , with B S O p r e t r e a t m e n t there was o n l y a m i n o r increase in c - D D P - i n d u c e d r e n a l d a m a g e at 24 h after c - D D P inject i o n i n d i c a t i n g t h a t G S H status m a y p l a y o n l y a m i n o r role in the toxicity o f cDDP. Interestingly, at 12 a n d 24 h a f t e r c - D D P i n j e c t i o n in the k i d n e y a n d at 24 h in the liver, M T c o n c e n t r a t i o n s were significantly g r e a t e r in the B S O p r e t r e a t e d g r o u p as c o m p a r e d to the n o n - p r e t r e a t e d g r o u p ( T a b l e IV). A l t h o u g h the renal c o n c e n t r a t i o n s o f Z n were n o t c h a n g e d , t h e r e was a significant increase in the p r o p o r t i o n o f Z n a s s o c i a t e d with the M T f r a c t i o n in k i d n e y cytosol. T h e r e was no similar c h a n g e in the cytosolic d i s t r i b u t i o n o f P t which was still p r e d o m i n a n t l y b o u n d to p r o t e i n s o f H M W . T h e m e c h a n i s m s i n v o l v e d in this increase in M T c o n c e n t r a t i o n s u n d e r c o n d i t i o n s o f G S H d e p l e t i o n are n o t k n o w n . It is p o s sible t h a t the i n h i b i t i o n o f G S H synthesis after B S O t r e a t m e n t m a y result in an i n c r e a s e d a v a i l a b i l i t y o f i n t r a c e l l u l a r cysteine for M T synthesis. A c h a n g e in the d e g r a d a t i o n rate o f M T to m a i n t a i n the tissue t h i o l levels also c a n n o t be ruled o u t u n d e r these e x p e r i m e n t a l c o n d i t i o n s . F u r t h e r studies are r e q u i r e d to d e l i n e a t e the m e c h a n i s m s i n v o l v e d a n d the significance o f G S H in the m e t a b o l i s m o f M T in the kidney.
Acknowledgements T h e a u t h o r s wish t o t h a n k M r . R o n N o s e w o r t h y for s t a n d a r d i z i n g the F A A S m e t h o d o f analysis for P t . This w o r k was s u p p o r t e d b y g r a n t s f r o m the M e d i c a l Research Council of Canada.
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