Toxicology, 67 ( 1991 ) 155-- 164 Elsevier Scientific Publishers Ireland Ltd.

Effectiveness of N-acetylcysteine in protecting against mercuric chloride-induced nephrotoxicity Guillermina Girardi and Maria Monica Elias Farmacologia. Facultad de Ciencias Bioquimicas 3' Farmaceuticas. Universklad National de Rosario. Consejo National de lnvestigaeiones Cient(ficas y Tecnicas ( CON1CET). Cons~jo de lnvestigaciones de la Universidad National de Rosario ( CIUNR) (Argentina)

(Received August Ist, 1990; accepted December 15th, 1990)

Summary Mercuric chloride (HgCl2)-induced nephrotoxicity, as measured by functional and biochemical parameters was evaluated in rats at different kidney non-protein sulfhydryls (NPS) levels. Diethylmaleate (DEM) induced a 75% of NPS diminution 1 h after the administration. Renal function (clearance) and biochemical measurements (gamma-glutamyltranspeptidase activity in urine, and lipoperoxides in kidney tissue) were impaired when the animals were HgCIz-treated. Values were highly impaired when the kidneys were NPS-depleted and were improved when NPS pools were previously increased although they were not similar to control values. DEM treatment promoted a higher accumulation of HgC12 in both kidney and liver while NAC-treatment reduced significantly the metal content in these organs. These data are in favour of a positive relationship among mercury content and organ injury. On the other hand, mercury content increased while NPS levels diminished. NPS might play a role in the HgCI 2 detoxification and thus avoids mercury accumulation and mercury effects. Key wordw N-acetyl cysteine; Protection: Mercuric chloride: Nephrotoxicity

Introduction M e r c u r y has b e e n i d e n t i f i e d as a n i n d u c e r o f t o x i c m a n i f e s t a t i o n s such as i m p a i r m e n t o f electrolyte, w a t e r a n d n o n e l e c t r o l y t e t r a n s p o r t in a v a r i e t y o f cells a n d tissues [ 1 ] . T h e p r i n c i p a l t a r g e t o r g a n s for H g C l 2 - i n d u c e d t o x i c i t y are the k i d n e y s [ 2 ] . H o w e v e r , the f u n d a m e n t a l m e c h a n i s m ( s ) by w h i c h this m e t a l d a m a g e s cells has n o t b e e n identified. N o n - p r o t e i n s u l f h y d r y l s ( N P S ) m a y be d i r e c t l y i n v o l v e d in m e r c u r y e n t r y i n t o the t u b u l a r cells [ 2 ] . G l u t a t h i o n e ( G S H ) plays a crucial role in this p r o cess a n d its h i g h c o n c e n t r a t i o n in r e n a l tissue m a y e x p l a i n the a c c u m u l a t i o n o f the m e t a l in the k i d n e y [ 3 ] . W e h a v e p r e v i o u s l y d e s c r i b e d a p o s s i b l e synergistic effect Address all correspondence and reprint requests to." Dra. Ma. Monica Elias, Farmacologia, Facultad de Ciencias Bioquimicas y Farmaceuticas, Suipacha 570-2000 Rosario, Argentina.

0300-483x/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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between the direct effects of mercury and G S H renal deficiency, suggesting that renal G S H content is an important factor in the expression and progression of mercury nephrotoxicity [4]. Moreover, it has been described that G S H forms complexes with several heavy metals [ 5,6] and thus might function in protection of cells against metal toxicity. Since there is little information available on the mechanisms of G S H and/or NPS protection against mercury nephrotoxicity, we studied the renal function and some biochemical parameters associated with structural renal integrity and the NPS protective role in HgC12-treated rats which were pretreated with N-acetylcysteine (NAC) or which were GSH-depleted. Diethylmaleate (DEM) was used for GSH depletion. It depletes G S H by means of a conjugating reaction [ 7 ]. NAC has been shown to increase G S H levels in mammalian cells [8,9]. Materials and methods Animals and treatments

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 mantained on a standard diet and water ad libitum. The animals were food-deprived 12 h before the experiments but were allowed free access to water. The following experimental groups were studied: (i) control rats (C, n = 10); (ii) rats injected with a single dose of D E M (4.0 mmol/kg body wt i.p., DEM, n = 10), this D E M dose was reported as effective in depleting kidney G S H levels up to almost 25% of control values [ 10,11 ] ; (iii) rats injected with a single dose of HgC12 (5.0 mg/kg body wt s.c., Hg, n = 10, this HgCI_, dose was previously described as effective in developing renal maximal effects [4] ); (iv) rats injected with a single dose of N A C (2 mmol/kg body wt i.p., N A C was dissolved in saline and the response on renal NPS levels was previously assayed both in control and DEM-treated rats, 1 h (NAC, n = 8) and (NAC + DEM, n = 8) and 2 h (NAC 2, n = 8) and (NAC., + DEM, n = 8) after the N A C injection); (v) rats injected with D E M as described in (ii) and a single dose of HgC12 as in (iii) concomitantly (DEM + Hg, n = 10); (vi) rats injected with N A C (2 mmol/kg body wt i.p.) followed by a single dose of HgCI 2 as in (iii) 2 h later (NAC 2 + Hg, n = 10). All the experiments were begun at 1100 h and concluded between 1600 and 1700 h to minimize the influence of circadian variations. Clearance studies

The animals (except those described in i.v.) were subjected to clearance studies 1 h after the respective treatment. This time was selected from an analysis of renal G S H pool and renal functions after a single dose of D E M [ 10]. HgC12 nephrotoxicity was observed 1 h after injection [4,12,13]. The animals were anesthetized with urethane (1 g/kg body wt i.p.). The femoral vein and femoral artery were cannulated (P.E. 50) and a bladder catheter (3 m m i.d.) was inserted through a suprapubic incision. Animals were maintained in restraining cages throughout the experiments to facilitate urine collection. A solution containing mannitol (5 g%), inulin (1 gO/,,),p_ aminohippuric acid (PAH) (0.3 gO/,,) and lithium carbonate (5 mg%) was infused at a rate of 4.5 ml/h. Lithium clearance was used as a reliable measure of proximal reab-

156

sorption [ 14]. A 45-min e q u i l i b r a t i o n p e r i o d elapsed before clearance m e a s u r e m e n t s begun. T w o urine s a m p l e s were o b t a i n e d d u r i n g 30 min a n d b l o o d from the femoral a r t e r y was o b t a i n e d at the m i d p o i n t o f each period. A r t e r i a l b l o o d pressure was m e a s u r e d t h r o u g h o u t the e x p e r i m e n t s with a G o u l d S t a t h a m P23 D b t r a n s d u c e r and r e c o r d e d on a R i k a d e n k i recorder. G l o m e r u l a r filtration rate ( G F R ) was calculated from the inulin clearance, a n d the p l a s m a flow rate f r o m P A H clearance (CpAH). F r a c t i o n a l excretions o f w a t e r ( F E % H20), s o d i u m ( F E % Na), p o t a s s i u m ( F E % K), glucose ( F E % glu) a n d lithium ( F E % Li) were c a l c u l a t e d by c o n v e n t i o n a l f o r m u l a e for each animal. A t the end o f the experiments, the kidneys were removed and their G S H c o n t e n t was d e t e r m i n e d .

Biochemical assays K i d n e y s a n d livers from a n i m a l s b e l o n g i n g to all g r o u p s were o b t a i n e d at the end o f the clearance experiments. They were h o m o g e n i z e d in 1.15% w/v KCI. Lipid peroxi d a t i o n (LPO) was assayed as t h i o b a r b i t u r i c acid ( T B A ) reactive p r o d u c t s a c c o r d i n g to the m e t h o d o f O k h a w a et al. [ 15]. The u r i n a r y excretion rate o f g a m m a - g l u t a m y l transferase ( G G T ) also was determined. D a t a were expressed relative to the creatinine excretion rate.

Analysis of mercury distribution in kidneys and livers F o l l o w i n g the c l e a r a n c e e x p e r i m e n t s (2 h) HgC1 v D E M + Hg a n d N A C + Hg a n i m a l s were killed a n d the kidneys a n d liver were perfused with saline, excised and h o m o g e n i z e d . T o t a l H g c o n t e n t in every s a m p l e was d e t e r m i n e d a c c o r d i n g to J a c o b s a n d S i n g e r m a n [ 16]. TABLE I RENAL NPS LEVELS IN RESPONSE TO DIFFERENT TREATMENTS NPS (t~mol/g wet tissue) NAC (2 mmol/kg body wt, 1 h before)

NAC (2 mmol/kg body wt, 2 h before)

Control

1.64 .4- 0.01 (n = 10)

1.73 + 0.06 (n = 8)

3.43 -4- 0.15" (n = 81

DEM (4 mmol/kg body wt)

0.45 -4- 0.07 (n = 14)*

0.48 .4- 0.06 (n = 10~*

0.89 .4- 0.08*'~t (n = 101"*

HgCI2 (5 mg/kg body wt)

1.34 .4- 0.02 (n = 14)*

N.D.

2.02 + 0.08*'~ In = 12)***

*Statistically different from control (P < 0.05). **Statistically different from DEM-treated rats. (P < 0.05). ***Statistically different from HgCL-treated rats. (P < 0.051. OStatisticallydifferent from NAC-treated (2 h before) rats (P < 0.051. N.D., not determined

157

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Analytical procedures Inulin concentrations in serum and urine were determined by Roe's procedure [ 17J and PAH concentration in the samples were determined by Brun's method modified by Waugh and Beall (18). Sodium and potassium were measured by flame photometry, lithium by atomic absorption spectrophotometry. Glucose was determined in blood and urine by the o-toluidine method and creatinine in urine by Jaffe reaction (Wiener Lab, Rosario, Argentina). The urine volume was measured gravimetrically. G G T activity was assayed using gamma-L-glutamyl-p-nitroanilide as substrate. The increase in absorbance at 405 nm was recorded (GGT test cinetica Wiener Lab, Rosario, Argentina). Determination of renal NPS (non-protein sulfhydryls) was carried out in homogenates prepared in cold 5% trichloroacetic acid in 0.01 M HCI and measured as described by Ellman [19].

Statistical analysis Statistical analysis was performed by using an unpaired Student t-test; multiple comparisons were made by analysis of variance. P values less than or equal to 0.05 were considered statistically significant. Values are expressed as mean ± S.E.

Results

Kidney NPS concentrations following diJferent treatments Data are shown in Table I. Renal GSH concentration were reduced almost 75% 1 h after a single injection of DEM. As reported [4] HgCI 2 induced a small, but significant decrement in renal GSH levels. NAC treatment promoted an increase in renal NPS levels 2 h later and avoided NPS decrements due to DEM or HgCI_~. Nevertheless, these values remained lower than the respective control values.

Renal functional parameters Some renal functional parameters are shown in Fig. 1. CpAn presents a similar pattern to GFR; FE% H20 and FE% K are also similar to FE% Na and FE% glu varies as FE% Li. NAC pretreatment 2 h before the study did not modify renal function as compared with control values, in spite of the increment in renal NPS levels. On the other hand, NAC pretreatment improved or protected renal function in DEM and HgCIE-treated rats. Blood pressure did not change during the experiments and there were not differences between groups. As recently reported [4], greater damage by HgCi 2 can be observed when animals are GSH-depleted by DEM.

Urine GGT activity Urinary G G T activity, referred to creatinine excretion rate, measured in groups i, iii, v and vi is shown in Fig. 2. HgCL_ produced an increased G G T activity as expected from the acute nephrotoxic effects on the proximal tubules. Similarly to the alterations reported on renal functions, this value was statistically increased in DEM + HgCI2-treated animals and NAC pretreatment promoted a lower impairment, as compared with HgCl_,-rats.

159

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Fig. 2. GGT activity in urine and lipid peroxidation in kidney tissue of control, HgC12 + DEM- and HgCI 2 + NAC-treated rats. GTT/creatinine values in DEM-treated animals were 1.78 ± 0.3 I.U./mg and were statistically higher than control values (0.82 ± 0.06 l.U./mg). NAC-treatment not modified GGT/creatinine values. *Statistically different from control group. (P < 0.05). #Statistically different from HgC12-treated rats (P < 0.05). TABLE II LIPID P E R O X I D A T I O N ( M E A S U R E D BY TBA REACTIVE PRODUCTS (MDA nmol/g w.t.)) IN KIDNEY A N D LIVER Kidney Control (n = 6) HgCI 2 (n = 6) DEM (n = 6) NAC (n = 6) NAC 2 h (n = 6) DEM + HgCI 2 (n = 6) NAC 2 h + HgCI 2 (n = 9) NAC 2 h + DEM (n = 6)

278.6 + 25.7

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617.4 ± 55*

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397

± 25*

756 ± 42*

301

± 48

518 + 14

240.5 ± 20.7

483 ± 56

957.6 ± 60.8*

889 + 73*

206.3 ± 33.7**

413 ± 54**

240.5 + 20.7 #

448 ± 42 ~

*Statistically different from control values. (P < 0.01). **Statistically different from HgC12 values. (P < 0.01). #Statistically different from DEM values (P < 0.01).

160

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Lipoperoxidation estimation Data from kidney homogenates are shown in Fig. 2. LPO followed similar modifications to renal functions and urine GGT. Table II also collected LPO measured in the livers from the animals belonging to these experimental groups. Mercury distribution and excretion Kidney and liver mercury content are shown in Fig. 3. DEM-treatment promoted a higher accumulation of HgCI 2 in both organs while NAC treatment significantly reduced the contents of the metal. Discussion

In previous work [4], we have reported that a single dose of HgCI, causes acute renal damage in male rats. This damage was characterized by alterationsin renal function which were potentiated in DEM-treated animals. Consequently, renal GSH content was described as an important factor in the expression and progression of mercury nephrotoxicity. In the present study, we undertook a series of experiments to further define the role of cellular NPS depletion and augmentation in the protection against nephrotoxicity induced by acute HgC12 exposure. The agent used was DEM which depletes GSH by covalent binding [9]. NPS supplementation was done by administration of NAC. NAC has been shown to increase the level of GSH in mammalian cells [9]; it could be intracellularly deacetylated to yield cysteine and the cystein formed in this manner could then be rapidly utilized

161

for GSH synthesis [8]. We observed that NAC promotes a significant increase in renal NPS content 2 h after administration. Renal mercury effects were assessed not only through functional studies, but also by measuring urinary G G T activity. Renal G G T , an extrinsic brush border membrane protein, is considered an indicator of acute nephrotoxicity in rats [20]. Moreover, several studies have indicated the involvement of lipid peroxidation as a possible cause of HgCI 2 toxicity [21]. Accordingly lipid peroxides were assayed in control, DEM-treated and NAC-treated rats which received HgCI 2. With this experimental protocol we were able to assess the effects of GSH not only on functional effects associated with HgCI 2 but also on structural and biochemical effects in the proximal tubular cells. The results of the experiments reported herein indicate that the nephrotoxic effects of HgCI_~ (functional and biochemical) are either enhanced by GSH-depletion or prevented by GSH-augmentation. The data reinforce our previous conclusion that a synergistic effect between the direct effect of mercury and GSH renal deficiency might exist. Although we have only measured LPO levels, it is noticeable that the relationship between HgCI2-renal effects and renal GSH content could also be described, at least in part, in the liver. LPO was higher in the livers of DEM and HgCl2-treated animals than the values observed in control rats. Even though DEM-treatment diminished hepatic GSH, this diminution was not accompanied by higher values of LPO when the animals were also treated with HgCI 2. On the other hand, NAC pretreatment promoted LPO values almost similar to control values in the animals challenged with HgC12 (see Table II). The molecular role of GSH in the toxicity of certain metals has not yet been elucidated. A positive correlation of mercury content with GSH content in both liver and kidney has been described suggesting that there would be a role for GSH in mercury uptake by both organs [2]. This fact would allow us to predict protection of the liver and kidney from mercury effects when the animals were NPS-depleted. Since our data are not in accordance with this explanation (2), we studied mercury accumulation both in livers and kidneys of control, DEM and NAC-treated rats which received HgCi 2. Data favour a positive relationship amount mercury content and the organ injury. On the other hand, the mercury content increased while GSH levels diminished (Fig. 3). This pattern was observed both in kidney and liver. This difference between our data and those reported by Bagget et al. [2] and Richardson et al. [3] might be a consequence of the different strain of rats used and/or of the different time of administration of DEM and mercuric chloride. Another point to be taken in account is the colorimetric method used for the determination of mercury in tissue. Appropriate analytical procedures were carried out in order to assure complete recovery of mercury from tissue. Moreover, DEM does not interfere with the mercury analysis. On the other hand, several studies suggest that GSH may play a role in the detoxification of metals [23]. Another possibility might be that tissues are more sensitive to toxicant effects when their NPS levels are diminished and, inversely the improvement in NPS levels could promote cellular defense mechanisms [23]. The present study suggests that NPS might play an important role in promoting the HgCI z detoxification and in this way avoids mercury accumulation and mercury

162

effects. T h e d i r e c t e f f e c t s o f D E M

and NAC on the mercury distribution could not

be descarded due to the almost simultaneous administration

of both drugs.

Acknowledgements This work was supported by grants from Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Consejo de Investigaciones Universidad Nacional de R o s a r i o ( C I U N R ) a n d b y T W A S ( T h i r d W o r l d A c a d e m y o f Sciences) R e s e a r c h G r a n t T W A S R G B C 88-75. T h e a u t h o r s w i s h t o t h a n k t h e W i e n e r L a b . A r g e n t i n a f o r analytical equipment and kind support.

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