Effect of different renal glutathione levels on renal mercury disposition and excretion in the rat

Toxicology, 81 (1993) 57-67 Elsevier Scientific Publishers Ireland Ltd.

57

Effect of different renal glutathione levels on renal mercury disposition and excretion in the rat G u i l l e r m i n a G i r a r d i a n d M a r i a M 6 n i c a Elias Farmacologia, Facultad de Ciencias, Bioquimicas y Farmac(uticas, Universidad Nacional de Rosario, Consejo Nacional de Investigaciones, Cientificas y TOcnicas ( CONICET) - - Consejo de lnvestigaciones de la Universidad, Nacional de Rosario ( CIUNR) ( Rept~blica Argentina) (Received November 9th, 1992; accepted February 17th, 1993)

Summary Mercury renal disposition has been studied following HgCI 2 injection (5.0 mg/kg body wt., s.c.) in controls, diethylmaleate and N-acetylcysteine-treated rats. The different treatments were used to generate statistically different degrees of non-protein sulfhydryls concentration in kidneys. Diethylmaleate (4 mmol/kg body wt., i.p.) diminished kidney glutathione levels to 25% and N-acetylcysteine (2 mmol/kg body wt., i.p.) increased kidney non-protein sulfhydryls levels up to 75% compared with new controls. The amount of mercury in the kidneys, the mercury excretion rate in urine and the mercury plasma disappearance curves were calculated during 3 h post HgCl 2 injection. BUN was measured in plasma at the same time period to determine the onset of kidney damage. The results indicate a higher HgCl 2 renal clearance in N-acetylcysteine-treated rats compared to controls and less renal mercury accumulation. The data agree with diminished renal toxicity. On the other hand, renal mercury accumulation was higher and mercury renal clearance lower in diethylmaleate-treated animals, associated with higher renal toxicity. The results suggest that non-protein sulfhydryl levels (principally glutathione) might determine renal accumulation of mercury as well as its elimination rate and hence might enhance or mitigate the nephrotoxicity induced by the metal.

Key words: Mercury nephrotoxicity; Renal mercury disposition; Non-protein sulfhydryls; Renal mercury excretion

Introduction Mercury has been recognized as a highly toxic metal to man for many years. Inorganic mercury (HgC12) is predominantly accumulated in the renal cortex, and affects the morphology and function of the proximal tubules [I-4]. We have reported that renal glutathione (GSH) content is an important feature in the expression of mercury nephrotoxicity [5]. We found that reducing renal GSH by diethylmaleate (DEM) evoked a synergistic effect between the effects of mercury and GSH renal deficiency [5]. Moreover, the increase in the renal NPSH pool by N-acetylcysteine Correspondence to: Dra. M. M6nica Elias, Farmacologia, Facultad de Ciencias Bioquimicas y Farmac~uticas, Suipacha 570-2000 Rosario, Reprblica Argentina. 0300-483X/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

58 (NAC) protects against HgCl2-induced nephrotoxicity [6]. We could also observe that DEM-treatment promoted an increased accumulation of mercury in the kidney while NAC-treatment was effective in decreasing its concentration [6]. However, these observations are contrary to other reports [7-9] which stated that GSH depletion selectively reduces mercury accumulation within the kidney, although mortality following mercuric chloride is increased [8,10]. On the other hand, several works have described potent mercury detoxificating agents that are SH-containing compounds such as N-acetyl-DL-penicillamine [l 1], D-penicillamine [12] and sodium Nbenzyl-D-glucamine dithiocarbamate [13]. The present study was undertaken to evaluate the renal distribution and renal excretion of mercury at different levels of NPSH in the kidney. Different levels of NPSH were obtained by DEM and NAC in order to decrease or increase NPSH tissue concentration, respectively. Material and methods

Animals and treatment Adult male Wistar rats (300-350 g) were used in all experiments. They were kept two per cage and housed in rooms with controlled temperature (21-23°C), humidity and light (0600-1800 h). They were maintained on a standard diet and water ad libitum. The following experimental groups were studied: (1) rats injected with a single dose of HgCI2 (5.0 mg/kg body wt., s.c.) (control). This HgC12 dose was previously described as effective in producing maximum renal effects 1 h after injection [5]; (ii) rats injected with a single dose of DEM (4.0 mmol/kg body wt., i.p.; 1 h before the experiment) and a single dose of HgCI2 as described above concomitantly (Hg + DEM). This DEM dose is effective in depleting kidney GSH levels to almost 25% of control values [14,15]; (iii) rats injected with NAC (2 mmol/kg body wt., i.p.) followed by a single dose of HgCI2 (as in (i)) 2 h later (Hg + NAC). NAC treatment increased renal NPSH levels by 75% compared to control values 2 h post NAC-administration [6]; and (iv) to compare functional effects of mercury in the different groups (i-iii), blood urea nitrogen (BUN) and renal NPSH content in rats without mercury administration (untreated rats) were also measured. Previous reports from our laboratory indicated that BUN modifications were always parallel to kidney damage measured by clearance techniques in HgCl2-treated rats [5]. All experiments were begun at 1100 h and concluded between 1600-1700 h to minimize the influence of circadian variations. Experimental procedure The animals were anesthetized with sodium thiopental (70 mg/kg body wt., i.p.). The femoral vein and femoral artery were cannulated (PE 50) and a bladder catheter (3 mm, i.d.) was inserted through a suprapubic incision. A solution containing Dmanitol (5 g%) was infused at a rate of 4.5 ml/h. Urine samples were obtained during 30 min and blood from the femoral artery at the end of each 30-min period. After 3 h the animals were killed and exanguinated. The kidneys were promptly removed and their NPSH content were determined. Some animals, prepared as described, were killed either after 30 min, 1 h, 1.5, 2

59 or 3 h to measure Hg content in kidneys at these times after HgCI 2 injection. Urine and blood samples were obtained similarly as described above. Mercury concentration was determined in blood, urine, and kidney homogenates. Urinary Hg excretion rate was calculated using the urine flow by conventional formulae.

Analytical methods Determination of renal NPSH (principally GSH at least in control and DEM + Hg group) was carried out in homogenates prepared in cold 5% trichloroacetic acid in 0.01 M HCI and measured as described by Ellman [16]. Mercury content in plasma, urine and kidney homogenates was determined according to Jacobs and Singerman [17]. Appropriate calibration-curves were obtained with tissue homogenates, plasma and urine samples (with or without the addition of DEM) plus known amounts of mercuric chloride in order to assure the adequacy of the colorimetric method. BUN was determined spectrophotometrically (Berthelot method) with a commercial reagent kit (Wiener Lab., Argentina). Pharmacokinetic studies Pharmacokinetic parameters describing the disposition of mercury during the experimental time were calculated using a one-compartmental open model. Plasma disappearance rates were derived from the concentration from the peak value (assuming absorption is completed). The area under the curves (AUC) was calculated by the trapezoidal rule from time 0 to 3 h. Mercury renal clearance was calculated from the ratio of cumulative urine excretion and AUC. Statistical analysis Statistical analysis was done by using analysis of variance followed by Student's t-test. A probability of less than or equal to 0.05 was assumed to denote a significant difference. Values are expressed as mean ± S.E.M. Results

Mercury plasma concentrations after different treatments Data are presented in Fig. 1. Control animals showed maximal mercury plasma levels at 30 min post s.c. injection. DEM treatment delayed the maximal mercury plasma concentration appearance. This level was higher in (Hg + DEM)-treated rats than in controls. Mercury plasma concentrations reached lower values in (Hg + NAC)-treated rats. The kinetic constants of plasma mercury disappearance as shown in Table I. The data could suggest that the different treatments might modify the absorption process (at least in DEM-treatment), the accumulation by tissues and/or the elimination rates. Renal N P S H level, mercury content and mercury elimination by kidney The different treatments resulted in different renal NPSH levels; untreated rats: 2.0 ± 0.3 t~mol/g w.t. (n = 8); DEM-treated rats: 0.45 4- 0.05/~mol/g w.t. (n = 8) and

60

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Time (min) Fig. 1. Effects of different NPSH levels on plasma Hg 2+ concentration after a single dose of HgCI 2 (5.0 mg/kg body wt., s.c.). Data represent the mean ~- S.E.M. When S.E.M. values are absent, they are included in the symbol size. The insert represents regression lines obtained from every individual values assuming monoexponential decay function. Each point represents the mean value. The procedure was described in Table I. O Control-rats (n = 5); • DEM-treated rats (n = 5); A NAC-treated rats (n = 7). *Significantly different from control rats, P < 0.05.

NAC-treated rats, 2 h after NAC administration: c u r y a d m i n i s t r a t i o n c a u s e d , in all a n i m a l s , a renal-NPSH content compared to the respective was of the same magnitude during 30-120 min v e r s e a t a p p r o x i m a t e l y 3 h (Fig. 2).

3.4 ± 0.1 g m o l / g w.t. (n = 8). M e r s t a t i s t i c a l l y s i g n i f i c a n t d e c r e a s e in g r o u p w i t h o u t Hg. T h i s d i m i n u t i o n p o s t H g i n j e c t i o n a n d b e g a n t o re-

TABLE I PHARMACOKINETIC PARAMETERS DESCRIBING HgCI 2 DISPOSITION IN CONTROL, DEM- AND NAC-TREATED RATS The samples used for calculations are represented in Figure I. k values were obtained with every mercury plasma concentration measured from 60 rain post HgCI 2 injection in every group of animals. The points were fitted to a single exponential decay function. Similar procedure was performed for AUC calculations which were determined by Trapezoidal's rule.

Control (n -- 5) DEM (n = 5) NAC (n = 7)

AUC (gg. ml -I -min)

k (h -I)

1944 ± 62 2238 4- 114 1104 4- 45*

0.336 + 0.024 0.684 + 0.036* 0.722 ± 0.027*

• Statistically different from control rats, P < 0.05.

61

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180 Time

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Fig. 2. Effects of different treatments and HgCI 2 on renal NPSH levels. The values represent the mean ~ S.E.M. 0 rain represents control values, DEM-treated rats (4 mmol/kg body wt., i.p., 1 h after DEM-injection) and NAC-treated rats (2 mmol/kg body wt., i.p., 2 h after N A C injection). Each value was the mean of five animals for control and DEM-treated rats and seven for NAC-treated rats. 30-120 min represents data collected at 30, 60, 90 and 120 min post mercury injection, which were not statistically different but were different from the respective untreated group (without mercury) (P < 0.05). n = 5 for control and DEM-group and n = 7 for NAC-treated group. 180 min represents renal GSH values at this period which were statistically different from 30 to 120 values (P < 0.05) and from zero values (P < 0.05). Every group consisted of five animals, except the NAC-group that consisted of seven animals. [] Controlrats; [] DEM-treated rats; ml NAC-treated rats. *Significantly different from control rats at the same time post mercury injection (P < 0.05). #Significantly different from the respective untreated-rats (without mercury) (P < 0.05).

TABLE II K I D N E Y M E R C U R Y C O N T E N T A N D U R I N E M E R C U R Y R E C O V E R Y EXPRESSED AS DOSE PERCENTAGE AND RENAL MERCURY CLEARANCE Mercury (% dose)

Control (n = 5) DEM (n = 5) NAC (n = 7)

Hg renal CI (ml/min/kg body wt.)

Kidney

Urine

18.2 ~ 0.5

0.26 ± 0.03

0.39 4- 0.02

49.7 4- 9.0

0.19 4- 0.02*

0.29 ± 0.02*

5.6 4- 0.3

0.32 4- 0.01"

0.76 4- 0.03*

Values were calculated at 180 rain post mercury administration, n represents the number of animals in each group. *Statistically different from control rats, P < 0.05.

62

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Time (rain) Fig. 3, Renal disposition of HgCI2 during 3 h post HgCI2 administration. A: Renal mercury content. Data represent the mean • S.E.M. (When S.E.M. values are absent they are included in the symbol size.) • Represents significantlydifferent from control-rats values at the same time post mercury injection (P < 0.05). O Control-rats (n = 5); • DEM-treated rats (n = 5); A NAC-treated rats (n = 7). B: Urine mercury excretion rate. Data represent the mean ± S.E.M. *Represents significantly different from control-rats values at the same time post mercury injection (P < 0.05). O Control-rats (n = 5); • DEM-treated rats (n = 5); A NAC-treated rats (n = 7).

Mercury c o n t e n t in renal tissue presented a trend to m a i n t a i n the a c c u m u l a t i o n after 30 m i n post injection both in control a n d (Hg + D E M ) - t r e a t e d rats. (Hg + D E M ) - t r e a t e d a n i m a l s a c c u m u l a t e d higher a m o u n t s of the metal, while kidneys of (Hg + N A C ) - t r e a t e d rats stored similar a m o u n t s of Hg to control rats. This was observed d u r i n g the first 90 m i n a n d then the values diminished statistically with respect to control rats (Fig. 3A). A n inverse relationship was observed a m o n g renal N P S H c o n t e n t a n d renal Hg a c c u m u l a t i o n . Mercury urine excretion load (EL) in (Hg + D E M ) - r a t s was lower than in control rats while (Hg + N A C ) - r a t s mercury EL was highest d u r i n g the first 30 m i n post in-

63 TABLE III TIME COURSE IN THE BUN LEVELS (mg/dl) OF RATS AFTER DIFFERENT TREATMENTS Time (min)

Untreated-rats (n = 5) Control-rats (n = 5) DEM-rats (n = 5) NAC-rats (n = 7)

30

60

90

120

180

27 + 5

27 + 4

29 + 5

28.5 q- 3

29 + 8

51 ± 2*

79 ± 5*

79 ± 5*

66 + 5*

68 ± 3*

62 ± 7"**

91 a- 7"***

87 ± 7*

73 ± 7*

82 ± 6"**

44 ± 5*'**

31 + 4**

18 ± 5**

17 ± 3**

27 ± 5**

Number of animals for each group is reported in parentheses. Data are expressed as mean ± S.E.M. *Statistically different from untreated-rats values, P < 0.05. **Statistically different from control rats, P < 0.05.

jection and then decreased at every time tested till 180 min. Mercury urine recovery and renal mercury clearance calculated by E L / A U C were presented in Table II.

Effects of HgCI; on BUN after different treatments Control rats developed acute renal failure characterized by a significant increase in BUN. Kidney failure was detected at 30 rain post injection and persisted during the experiment without any changes. (Hg + DEM)-treated rats presented higher B U N than control rats while (Hg + NAC)-animals only presented a transient increment in B U N at 30 min post mercury injection (Table III).

Discussion The data presented showed that renal N P S H levels in rats (roughly representing G S H [18]) significantly affected excretion and kidney disposition o f mercury following subcutaneous administration o f HgCI 2. The data provide further information about the possible mechanisms involved in the potentiation and protection performed by N P S H against HgC12-kidney injury as previously reported [5,6]. It has been described that 2 days following s.c. administration o f 2.5/~mol/kg b o d y wt. HgCI 2 to rats, the whole-body mercury retention was 76% [19]. This long time period for mercury clearance was also described using different administration routes and different animal species [20-23]. Nevertheless, the present study was done after a short time following HgCI2 injection. It seemed important to describe mercury disposition during this time period due to the previous results on: (a) the acute renal failure in HgCl2-treated rats developed at this time [5]; and (b) the G S H depletion-potentiation [6] and N A C - p r o t e c t i o n [6] was observed at this early stage. Nevertheless, this period might belong to the distribution period in the HgCI2toxicokinetics.

64 The animals used in these experiments developed an acute renal failure due to mercury injection following similar patterns to those described by clearance techniques [5,6]. Different tissue NPSH levels were observed in kidneys in DEM- and NAC-treated rats and were modified by HgCI2 injection. It seems important to note that renal NPSH content was further depleted between 30 and 120 min post mercury injection while urine mercury excretion rate was maximal during this period in every experimental group; thus, urine mercury excretion could explain the renal NPSHdepletion after mercury administration. It has been reported that mercury liver excretion is determined primarily by forming metal GSH complexes [24]. Other works [25-27] described a direct involvement of GSH in the urinary excretion of mercury. Plasma Hg concentrations were lower in the NAC group than in control rats. This fact could be explained by a slower absorption process from the administration site (s.c.) and/or a higher elimination rate in NAC-treated rats than in controls. Endo et al. [28] described that chelating agents, such as cysteine, decrease HgCI2 absorption through the brush border membrane of small intestine. Nielsen et al. [29] found decreased whole-body mercury levels in rats exposed to intravenous mercuric chloride and injected with thiol-containing chelators such as 2,3-dimercaptosuccinic acid (DMSA) and 2,3-dimercaptopropano-l-supphonate (DMPS). Both reports support a diminished Hg transport across membranes in the presence of chelating agents. In the present work mercury was injected s.c. 2 h after NAC (i.p.). It was observed that mercury plasma levels at 30 min post mercury injection were similar in DEMand NAC-treated rats despite their different NPSH tissue content, which might determine different systemic NPSH levels. Thus, differences in the absorption process appear unlikely. On the other hand, we described that plasma mercury concentration decay and urine mercury elimination rate in NAC-treated rats were higher than in control animals and which were similar to data reported by Nielsen et al. [29]. Therefore, our data favor the higher elimination rate hypothesis in NAC-treated rats. Moreover, Hg kidney contents were almost similar in control and NAC-treated rats during the first periods; later, NAC-treated rats had a lower metal content. It is possible that NPSH blood levels were higher in NAC-treated animals, thus HgCI2 could be highly chelated and metal tissue renal uptake could be impaired [28]. These results are different from data reported by Bagget and Berndt [7], who found a positive correlation between mercury content and NPSH in kidney. It is remarkable that these authors reported this relationship only under NPSH-depleted conditions. Moreover, the increase in renal GSH following GSH monoethylester was not associated with an increase in renal HgCI2 accumulation [30]. The higher mercury excretion rate and the diminished mercury renal accumulation (see Table II) could explain the protection against mercury salt damage observed in our laboratory [6] during NAC pretreatment and also described in this paper (Table III). It has been reported that the critical cellular events that ultimately lead to acute HgCIE-nephrotoxicity probably occur within the first hours following exposure [31,32]. It seems likely that the diminished mercury content seen within the first hours is the most critical. The plasma disappearance of mercury is higher in DEM-treated animals than in

65 controls. This could be explained by the higher accumulation of Hg in kidney and other tissues (data not shown) compared with control animals. In the first periods studied, the higher kidney content was not associated with higher urine Hg elimination. Moreover, mercury renal clearance in DEM-treated rats was diminished as compared with control-rats. It is noticeable that the higher metal content in DEM-treated rats was observed in spite of lower NPSH levels than values measured in control kidneys. These observations, together with the findings made in NAC-rats, might be considered in contrast with data reported by Baggett and Berndt [7]. They used a similar experimental protocol but a different dose of mercury, different time intervals between DEM and HgC12 administration and different strains of rats. These facts could account, in part, for the discrepancy. Nevertheless, similar results were reported by others [8-9]. It is noticeable that Bagget and Berndt also demonstrated a lack of an effect in mercury uptake in NPSH-depleted slices compared to slices from control rats [7]. Moreover, mercury accumulation within the renal cortex was found to be increased in rats with unilateral nephrectomy while tissue sulfhydryl content was not altered [32]. It seems thus possible that NPSH could be an important, although not the only, determinant of mercury accumulation. It is likely that we are describing the result of mercury uptake process in DEM-rats similar to control values (as reported in renal slices [7]) or an increased uptake process of Hg(GSH)2 by a rglutamyltranspeptidase dependent transport system. It has been described that the chemical form of urinary mercury in mice is Hg(GSH)2 [26]. After DEM treatment, GSH repletion rates observed in the kidney were higher than in liver [16] principally from GSH-derivatives by r-glutamyltranspeptidase [33]. Whatever the actual value of the uptake process was, the diminished intracellular renal NPSH pool might have favoured mercury tissue accumulation in other structures (membranes, proteins) avoiding the elimination in urine, contrary to the effect observed in NAC-treated rats (see Fig. 3A). Altogether these possibilities could explain the higher renal mercury content compared with control animals. At present, these extremely divergent observations on the role of tissue sulfhydrils in the HgCI 2 accumulation process remain unexplained and need further experiments which allow dissection of the uptake from the efflux processes. Regarding the issue of renal GSH content and HgC12 nephrotoxicity, our results agree with those reported on GSH-depletion potentiated HgC12 nephrotoxicity [8-10], although Johnson [9] described that GSH-depletion preserved renal function following HgCI 2. This work [9] described diminished renal mercury content but also similar glomerular filtration rate in all the experimental groups (DEM, DEM + Hg and Hg). We have found that renal function in (DEM + Hg)-treated rats were impaired as compared with the DEM-group at every mercury dose and time period assayed [5]. On the other hand, different treatments promoting increased NPSH renal levels ameliorate the nephrotoxicity of HgC12 [6,30,34-35] as described in this work. These data on DEM-treated rats point to the possibility that GSH depletion may increase the susceptibility of animals to the toxicity of mercury, favouring HgCI2 accumulation in the kidney, but leaving it unsequestered, making it available for toxic interactions with other sensitive ligands within the cells. On the other hand,

66

higher NPSH levels could maintain mercury sequestered and thus favor its excretion and avoid its toxic effects.

Acknowledgements This work was supported by the Consejo Nacional de Investigaciones Cientificas y Trcnicas (CONICET). Reptiblica Argentina and by a Third World Academy of Sciences (TWAS), Italy, Research Grant (TWAS RG BC 88-75). The authors thank Wiener Lab. Rosario, Argentina for the gift of analytical reagents.

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