Pretreatment with p-aminohippurate inhibits the renal uptake and accumulation of injected inorganic mercury in the rat

Transcription

1 ELSEVIER Toxicology 103 (1995) Pretreatment with p-aminohippurate inhibits the renal uptake and accumulation of injected inorganic mercury in the rat Rudolfs K. Zalups* a, Delon W. Barfussb Division of Basic Medical Sciences, Mercer University School of Medicine, 1550 College Street, &acon. GA 31207, USA bdepartment of Biology, Georgia State University, Atlanta, GA 30303, USA Received 1 February 1995; accepted 16 March 1995 AbShCt The effects of intravenous pretreatment with the organic anion, p-aminohippurate (PAH) on the disposition of intravenously administered inorganic mercury in the kidneys, liver and blood were evaluated in rats. In dose-response experiments, the renal uptake (and/or accumulation) of mercury, 1 h after the injection of a nontoxic 0.5 pmol/kg dose of mercuric chloride (HgClJ, was significantly reduced in rats when a 1.0,3.3 or IO mmok kg dose of PAH was administered 5 mitt prior to the injection of HgCl,. This reduction was due to reduced uptake of mercury in both the renal cortex and outer stripe of the outer medulla. Near maximal inhibition appeared to be achieved with the 10 mmol/kg dose of PA.H. Inhibition of the uptake (and/or accumulation) of mercury in the renal cortex and outer stripe of the outer medulla, 1 h after the injection of the nontoxic dose of HgClr, was also detected in experiments where HgC!l, was injected!i,30,60 or 180 min after pretreatment with a 10 mmolikg dose of PAH. The renal uptake of mercury was inhibited significantly when the nontoxic dose of inorganic mercury was administered 5, 30 or 60, but not 180 min after pretreatment with the 10 mmol/kg dose of PAH. In another experiment, the renal burden of mercury was significantly reduced for 24 h when pretreatment with a 10 mmoykg dose of PAH was administered 5 min prior to the injection of HgCl,. Pretreatment with PAH did not have an effect on the hepatic disposition of mercury, but it did cause a significant increase in the fraction of mercury present in the plasma of blood. In summary, the findings in the present study indicate that pretreatment with PAH inhibits the renal uptake of injected inorganic mercury in a dose-dependent and timedependent manner. In addition, the findings tend to indicate that some fraction of the mercury that enters into renal tubular epithelial cells is by a mechanism involving the organic anion transport system. Keyworrlr: Renal uptake ; Inorganic mercury ; p-aminohippurate ; Organic anion transport system 1. Intraluction The renal uptake, accumulation (Tanaka et al., 1992) and toxicity (Ban and de Ceaurriz, 1988) of methylmercury have been shown to be reduced Ccncsponding author, Tel.: (912) ; Fax: (912) significantly in mice pretreated with the organic anion probenecid, which is a well-characterized corripetitive inhibitor of the renal uptake and transport of other organic anions (Roth-Ramel et al., 1992). Thus, these findings tend to implicate the renal organic anion transport system in some aspect of the renal tubular uptake of organic mercurie compounds Elsevier Science Ireland Ltd. All rights reserved SSDI X(95)

2 24 RX. Zalups. D. W. Barfiiss / Toxicology 103 (1995) It is unclear, however, whether pretreatment with an organic anion affects the renal uptake and disposition of inorganic mercury. Therefore, one of the principal aims of the present study was to test the hypothesis that pretreatment with an organic anion, that competitively inhibits the renal tubular transport of other organic anions and that does not contain a free sulfhydryl group with which a mercuric can bind, causes a significant reduction in the renal tubular uptake of administered inorganic mercury. This hypothesis was tested in three separate experiments. In the first experiment, the shortterm (1 h) renal and hepatic disposition of inorganic mercury was evaluated in rats pretreated, 5 min prior to the intravenous injection of a nontoxic dose of mercuric chloride, with varying doses of the water-soluble organic anion p-aminohippurate (PAH). This organic anion, like probenecid, is a well-established competitive inhibitor of proximal tubular transport of a number of other organic anions (Roth-Ramel et al., 1992). In a second experiment, the short-term renal and hepatic disposition of inorganic mercury was evaluated in rats pretreated with a near maximal dose of PAH at varying times prior to the administration of mercury. The third experiment was designed to evaluate the effect of pretreatment with a near maximal dose of PAH on the renal and hepatic disposition of inorganic mercury 24 h after the administration of mercuric chloride Animals Male Sprague-Dawley rats were used in the present study. They were purchased from Harlan Sprague-Dawley (Indianapolis, IN) at a weight of g. All animals were allowed at least several days of acclimation prior to any experimentation. Water and a commercial laboratory diet for rats were provided ad libitum during all phases of the study. renal uptake and/or accumulation of injected inorganic mercury. In the first experiment (experiment 1), the effects of pretreatment-dose of PAH on the disposition of mercury in the kidneys, liver and blood were evaluated in rats 1 h after they were injected intravenously with a nontoxic dose (0.5 pmol/kg) of mercuric chloride (HgC12). In the second experiment (experiment 2), the effect of time of pretreatment with PAH on the disposition of mercury in the kidneys, liver and blood was evaluated in rats 1 h after they were injected intravenously with the nontoxic dose of HgCl,. In the last experiment (experiment 3), the disposition of mercury in the kidneys, liver and blood, and the urinary and fecal excretion of mercury were evaluated 24 h after rats pretreated with PAH were injected with the nontoxic dose of HgC Groups and treatments Experiment 1. Four groups of four rats were used in experiment 1. All four groups of rats received some form of pretreatment 5 min prior to the administration of an intravenous, 0.5 pmol/kg, nontoxic dose of HgC12. Each pretreatment was administered into the right femoral vein in a vehicle consisting of 0.9% (w/v) aqueous sodium chloride at a volume to body weight ratio of 4.0 ml/kg. One group of rats received only the saline vehicle as a pretreatment and served as a control. The remaining three groups of rats received varying doses of p-aminohippurate (PAH). One group received a 1 mmol/kg dose of PAH, a second 2. Materials and methods group received a 3.3 rnmol/kg dose of PAH and the fourth group received a 10 mmol/kg dose of PAH. All animals in each of the four groups were administered the 0.5 pmol/kg dose of HgClz into the left femoral vein, in 2.0 rni/kg 0.9% aqueous sodium chloride, 5 min subsequent to the intravenous administration of the designated pretreatment. The injection-solution used to administer the 0.5 pmol/kg dose of HgCl2 also contained 5 &i/ml of [203Hg]C12(Buffalo Materials Corp., Buffalo, NY), which had a specific activity of mci/mg 2.2. Experiments at the time of experimentation. Three separate experiments were carried out to All the animals in each of the four groups were test the hypothesis that the organic anion p- anesthetized with a 100 mg/kg intraperitoneal dose aminohippurate (PAH) inhibits or diminishes the of sodium pentobarbital 1 h after the administra-

3 R K. Zalups, D. W. Bofis / Toximlogy 103 (1995) tion of the 0.5 mol/kg dose of HgC12. Once anesthesia was induced, samples of blood, liver and renal tissues were obtained Experim ent 2. Eight groups of 4-5 rats were used in this experiment. Four of the groups were pretreated with a 10 mmovkg intravenous dose of PAH (a.dministered in 4.0 ml/kg 0.9% aqueous sodium chloride) and the other four groups were pretreated with vehicle only (4.0 ml/kg). Pretreatment-injections were administered into the right femoral vein. One group of rats pretreated with PAH and one group of control rats pretreated with sabne received an intravenous, 0.5 clmovkg dose of HgCl, into the left femoral vein 5 min after pretreatment. Another set of paired groups of PAHpretreated and saline-pretreated rats received the dose of HgCl, 30 min after pretreatment. The third and fourth sets of paired groups received the dose of HgC& 60 and 180 min after pretreatment, respectively. All animals were anesthetized with ;B 100 mg/kg intraperitoneal dose of sodium pentobiarbital 1 h after the intravenous dose of HgClz and then samples of blood, liver and renal tissue were obtained Experiment 3. Two groups of five rats were used in this experiment. One of the groups was pretreated with a 10 mmol/kg intravenous dose of PAH (in 4.0 ml/kg 0.9% aqueous sodium chloride) and the other group was pretreated with vehicle only (4.0 ml/kg). The pretreatments were administered into the right femoral vein. Both groups of rats were administered a 0.5 pmollkg intravenous dose of HgC12 into the left femoral vein 5 min after pretreatment. After the administration of HgCl*, each animal was placed individually in a plastic metabolic cage for 24 h. At the end of the 24-h experimental period, samples of blood, liver and renal tissue were obtained from the animals, after they were anesthetized with a 100 mg/kg intraperitoneal dose of sodium pentobarbital Procedure of intravenous injections. Each animal was anesthetized lightly with ether prior to each of the two intravenous injections. Once anesthesia was achieved, a small incision was made, with a small pair surgical scissors, through the skin in the mid-ventral region of the thigh to expose the femoral vein in which the injection was to be administered. After the fascia around the femoral vein was trimmed, the designated injection-solution was administered into the vein using a 1 ml syringe equipped with a 5/8 inch 25- gauge needle. Afterwards, the opposite ends of the incised skin were approximated using a couple of sterile 9 mm surgical wound clips Collection and handling of urine and feces All urine and feces excreted in 24 h by each animal in experiment 3 were collected. At the end of the 24-h collection period, the total amounts of urine and feces excreted were weighed. The entire amount of feces excreted during the 24-h period was placed and sealed in 16 x 95 mm polypropylene y-counting tubes. From the total volume of urine excreted during the 24-h period, a 1.0 ml sample was obtained and then placed and sealed in a 12 x 75 mm polystyrene y-counting tube. The amount of mercury excreted in the feces and urine by each animal was determined by standard ^/- counting techniques Collection of tissues and organs After each animal in the present study was anesthetized with sodium pentobarbital, two 1 ml samples of blood were obtained from the inferior vena cava with a 10 ml syringe and a 20-gauge needle. One of the samples was placed directly into a 12 x 75 mm polystyrene y-counting tube. The other sample was placed in a 1.5 ml polypropylene microcentrifuge tube and centrifuged at 3000 x g for 7 min. Subsequently, the plasma fraction was separated from cellular fraction. Each fraction was placed individually in a tared polystyrene y- counting tube. Separation of the two fractions of blood allowed for the determination of the distribution of mercury between the plasma and the cellular fractions of blood. After the samples of blood were drawn, the kidney(s) and liver were excised and weighed quickly. The left kidney was sliced in half along the transverse plain. One of the halves was place in a tared r-counting tube. A 3-mm transverse slice of kidney was obtained from the other half. Samples of cortex, outer stripe of the outer medulla, inner stripe of the inner medulla and inner medulla were obtained from this slice of kidney by careful dissec-

4 26 R.K. Zalups. D. W. Barfwss/ Toxicology 103 (1995) tion. A l-g sample of liver was also obtained. All samples of renal and hepatic tissues were placed and sealed in tared 12 x 75 mm y-counting tubes Determination of the content of mercury in samples of tissue, urine and feces The radioactivity of *03Hg in the samples of tissues, organs, urine, feces and injection solutions was determined by counting the samples in a 1282 Compugamma CS deep-well gamma spectrometer (Pharmacia-LKB, Gaithersburg, MD) operating at a counting efficiency of 500/o for *03Hg. The actual content of inorganic mercury (Hg++) in each sample was calculated by dividing the radioactivity of *03Hg in the sample (dpm) by the specific activity of *03Hg in the injection solution (dpmnmol). The concentration of inorganic mercury in the samples of tissues is expressed as percent of the administered dose per g of tissue. The total content of mercury in the whole liver and kidney(s) is expressed simply as percent of the administered dose. Urinary excretion of mercury and fecal excretion of mercury are expressed as percent of the administered dose excreted in 24 h Statistics All values are expressed as mean SE. Differences between means for individual sets of data obtained from the groups of rats in experiment 1 were evaluated statistically by first using a 1 x 4 one-way analysis of variance (ANOVA). When F- values obtained with the ANOVA were found to be statistically significant (P < 0.05) the Tukey s protected-t post-hoc multiple comparison test was used to determined which means were significantly different from one another. In experiment 2 and 3, differences between means for individual sets of data obtained from corresponding paired groups of PAH-pretreated and saline-pretreated rats were evaluated statistically by using the unpaired Student s t-test for independent samples. All scores expressed as a percent of the dose of mercury in the present study were transformed by the arc-sine transformation prior to performing any statistical analyses. This was done because data expressed as a percent, or fraction of some total value, do not generally fit a normal or Gaus- Sian distribution. The arc-sine transformation normalizes percent data by taking the arc-sine of the square root of n, where n is the decimal fraction of the percent score. The level of significance (P c 0.05) for all statistical analyses performed is the present study was chosen a priori. 3. Results 3.1. Experiment Renal disposition of mercury. The concentration of mercury (% dose/g tissue) in the left kid- 13 J J,,,,,,,,,,,,, Dose of p_aminohippurate 5 Minutes Prior to injection of Hg++ (mmollkg) Fig. 1. Concentration of mercury (% dose/g tissue) in the left kidney and the content of mercury in the total renal mass, 1 h after the intravenous administration of a 0.5 mol/kg nontoxic dose of mercuric chloride (containing 203HgC1s), in rats pretreated with a 0, 1.0, 3.3 or 10 mmokkg dose of p arninohippurate (PAH) administered intravenously 5 mitt prior to the injection of mercuric chloride. Values represent mean SE. for four animals per group. *Significantly different (P < 0.05) from the mean for the control group of rats that did not receive PAH.

5 R.K. Zahps. D. W. Bcvfurs/ Toxieolm 103 (1995) a 20 E f 14 $ 13 ZJ o- 3% 12 ga.c 11 tjs. L- qj a 10 2 L 9 m I d,,,,, I I I I I1 I Dose of paminohippurate 5 Minutes Prior to Injection of Hgf+ (mmollkg) ney (Fig. 1) and the content of mercury (% dose) in the total renal mass (Fig. 1) in each of the three groups of rats pretreated intravenously with PAH (1, 3.3 or 10 mmol/kg) were significantly lower than those in the group of control rats pretreated with normal saline. However, differences in the concentration of mercury in the left kidney or in the content of mercury in the total renal mass between the three groups of rats pretreated with PAH were found not to be statistically different Intrarenal distribution of mercury. Significant differences in the concentration of mercury (% dose/g tissue) were detected in the renal cortex and outer stripe of the outer medulla between the four groups of rats in this experiment. The renal cortical concentration of mercury was significantly lower in all three groups of rats pretreated with Fig. 2. Concentration of mercury (% dose/g tissue) in the renal cortex and outer stripe of the outer medulla, 1 h after the intravenous administration of a 0.5 molkg nontoxic dose of mercuric chloride (containing 203HgCl,), in rats pretreated with a 0, 1.O, 3.3 or 10 mmoukg dose of paminohippurate (PAH) administered intravenously 5 min prior to the injection of mercuric chloride. Values represent mean f SE. for four animals per group. *Significantly different (P c 0.05) from the mean for the control group of rats that did not receive PAH. Table 1 Effect of pretreatment dose of paminohippurate (PAH) on the disposition of injected inorganic mercury Type of pretreatment Time after Animal body [Hg] in the [Hgl in IM Content of Hg Content of Hg pretreat- weight (g) ISOM (% dose) in liver (% dose) in blood (% ment when (% dose/g) dose) Hg was injected (tin) Saline 5 227a6(n=4) 0.98 zt * * i 1.06 PAH (1 mmolkg) (n=4) 1.01 zt f 2.13 PAH (3.3 mmolikg) 5 222*4@=4) O.% f f * PAH (10 mmolkg) 5 222*3(n=4) 0.91 f f f ztz 0.81 Values are mean f SE. Both saline and PAH were administered into the right femoral vein (4.0 ml&g. At the end of the 5-min pretreatment period, a 01.5 WoUkg dose (in 2.0 mlkg) of mercuric chloride (containing 203HgCld was administered into the left femoral vein. Tissuea weire obtained from all the animals 1 h after the intravenous dose of mercuric chloride was administered. The total volume of blood in each animal was estimated to be 6% of weight of the animal. ISOM, inner stripe of the outer medulla. IM, inner medulla.

6 28 R.K. Zalups, D. W. Barfms / Toxicology 103 (1995) PAH than in the group of control rats (Fig. 2). Moreover, the renal cortical concentration of mercury in the rats pretreated with the 10 mmol/kg dose of PAH was also significantly less than that in the rats pretreated with 1.0 mmol/kg dose of PAH. In the groups of rats pretreated with the 3.3 or 10 mmol/kg dose of PAH, the concentration of mercury in the outer stripe of the outer medulla was significantly less than that in the outer stripe of the outer medulla of the control rats pretreated with saline only (Fig. 2). No other statistically significant differences, with the respect to the concentration of mercury in the outer stripe of the outer medulla, were detected. There were no statistically significant differences in concentration of mercury in either the inner stripe of the outer medulla or the inner medulla among the four groups of rats in experiment 1 (Table 1) Disposition of mercury in blood and liver. No significant differences were detected with respect to the content of mercury (Oh dose) in the blood or liver among the four groups of animals in this experiment (Table 1). There were, however, significant differences in the distribution of mercury between the cellular and plasma fractions of blood among the four groups of animals, par- $ 25 : a 20 J, t,,,,,,, ( (, ( Dose of p-aminohippurate 5 Minutes Prior to lnpction of Hg * (mmol/kg) Fig. 3. Percent of the mercury in blood present in the plasma, 1 h after the intravenous administration of a 0.5 pmolikg nontoxic dose of mercuric chloride (containing 203HgCl& in rats pretreated with a 0, 1.0, 3.3 or 10 mmoykg dose of p aminohippurate (PAH) administered intravenously 5 mitt prior to the injection of mercuric chloride. Values represent mean f SE. for four animals per group. *Significantly different (P < 0.05) from the mean for the control group of rats that did not receive PAH. 1, 30 5% ;s 25 & 20 I- % 15 E J&l 10 C s Ill. 30 min. 60 min. 180 ml Pretreatment Time Prior To Administration of Hg= Fig. 4. Concentration of mercury (% dose/g tissue) in the left kidney and the content of mercury in the total renal mass, 1 h after the intravenous administration of a 0.5 pmovkg nontoxic dose of mercuric chloride (containing 203HgC12), in rats pretreated intravenously with normal saline (09% aqueous NaCl) or a 10 mmol/kg dose of p-aminohippurate (PAH) 5, 30, 60 or 180 mitt prior to the injection of HgCl2. Values represent mean f S.E. for live animals per group, except for the groups of rats pretreated with saline 5 or 30 min prior to, or the group of rats pretreated with PAH 5 mitt prior to, the injection of the nontoxic dose of HgC12, in which there were four rats per group. *Significantly different (P c 0.05) from the mean for the corresponding group of control rats pretreated with normal saline.

7 R. K, Zulups. D., W. Barfius / Toxicology 103 (1995) titularly in the groups of rats pretreated with the higher doses of PAH (Fig. 3). Significantly mom mercury was detected in the plasma fraction of blood in the group of rats pretreated with the 3.3 mmoykg dose of PAH than in the group of control rats or the group of rats pretreated with the 1.0 mmolikg dose of PAH (Fig. 3). In addition, the amount of mercury in the plasma fraction of blood in the animals p:retreated with the 10 mmovkg dose of PAH was significantly greater than that in the rats of the other three groups Experiment Renal d&position of mercury. When the nontoxic 0.5 pmol/kg dose of HgC12 was administered at 5, 30 and 60 min after pretreatment, the concentration of mercury in the left kidney (Fig. 4) and the content of mercury in the total renal mass (Fig. 4) were signiticantly less in the rats pretreated with the 10 mmol/kg dose of PAH than in the corresponding rats pretreated with normal saline 1 h after the inorganic mercury was administered. There was, however, no statistically significant difference in the concentration of mercury in the left kidney, or in the content of mercury in the total renal mass, between the group of rats pretreated with PAH and the group of rats pretreated with saline that were administered the nontoxic dose of HgC min after pretreatment Intrarenai distribution of mercury. The renal cortical concentration of mercury was also significantly less in rats pretreated with the 10 mmohkg dose of PAH than in corresponding rats pretreated with sahe when the dose of HgC12 was administered 5,30 and 60, but not at 180 min, after pretreatment (Fig. 5). A significant difference in the concentration of mercury in the outer stripe of the outer medulla between rats pretreated with PAH and the rats pretreated with saline was detected only when the nontoxic dose of I+$& was administered 30 min after pretreatment (Fig. 5). In this case, the concentration of mercury in the outer stripe of the outer medulla was significantly less in the group of rats pretreated with PAH. There were no statistically significant differences in concentration of mercury in either the 5 min. 30 min. 60 min. 180 min. Pretreatment Time Prior To Administration of Hg- Fig. 5. Concentration of mercury (% do&g tissue) in the renal cortex and outer stripe of the outer medulla, 1 h after the intravenous administration of a 0.5 ~~~~ovltg nontoxic dose of mercuric chloride (containing 203HgCla, in rats pretreat& intravenously with normal saline (0.9% aqueous NaCI) or a 10 mm&kg dose of p-aminobippurate (PAH) 5,30,60 or 180 min prior to the injection of HgC12. Values represent mean f S.E. for five animal per group, except for the groups of rats pretceated with saline 5 or 30 min prior to, or the group of rats pretreated with PAH 5 min prior to, the injection of the nontoxic dose of HgC&, in which there were four rats per group. Signikantly different (P < 0.05) from the mean for the corresponding group of control rats pretreated with normal saline. inner stripe of the outer medulla or the inner medulla, 1 h after the intravenous injection of the nontoxic dose of HgC12, among the paired groups of rats studied 5,30,60 and 180 min after pretreatment with either PAH or saline (Table 2) Disposition of mercury in blood and liver. Siguificant differences in the content of mercury in the blood or liver were not detected (1 h after the injection of the nontoxic dose of HgCl& between

8 30 R.K. Zalups. D. W. Barfiss/ Toxicology IO3 (1995) Table 2 Effect of time after pretreatment with paminohippurate (PAH) on the disposition of injected inorganic mercury Type of pretreatment Time after Animal body [Hg] in the [Hg] in IM Content of Hg Content of Hg pretreat- weight (9) ISOM (% dose/g) in liver (% dose) in blood (% ment when (% dose/g) dose) Hg was injected (mm) Saline 5 PAH (IO mmolkg) 5 Saline 30 PAH (10 mmohkg) 30 Saline 60 PAH (10 mmolkg) 60 Saline 180 PAH (10 mmohkg) *2(n=4) 207 i 3 (n = 4) 204+3(n=4) (n = 5) 201 * (a = 5) (n = 5) (n = 5) 223 zk 3 (n = 5) 0.82 f o. 1 I 0.93 * f f * f zt zt zk f f i f f f zt f * zk f ZIZ f f f zt f zt zt 0.62 Values are mean f S.E. Both saline and PAH were administered into the right femoral vein (4.0 ml/kg). At the end of each pretrcatment period, a 0.5 pmolikg dose (in 2.0 ml/kg) of mercuric chloride (containing 203HgC12) was administered into the left femoral vein. Tissues were obtained from all the animals I h after the intravenous dose of mercuric chloride was administered. The total volume of blood in each animal was estimated to be 6% of weight of the animal. ISOM, inner stripe of the outer medulla. IM, inner medulla. any of the pairs of PAH-pretreated and salinepretreated rats in experiment 2 (Table 2). However, significant differences in the distribution of mercury in blood were found between rats pretreated with PAH and rats pretreated with saline when the animals were injected with HgCl* 5, 30 and 60 min, but not 180 min, after pretreatment (Fig. 6). When HgCl* was administered 5, 30 and 60 min after pretreatment, significantly more mercury was in the plasma fraction of blood in the rats pretreated with the 10 mmol/kg dose of PAH than in the rats pretreated with saline 1 h after the intravenous injection of HgClz Experiment Renal disposition of mercury. The concentration of mercury in the left kidney (Table 3) and the content of mercury in the left kidney and total renal mass (Fig. 7), 24 h after the injection the 0.5 PmoYkg dose of HgClr, were significantly lower Fig. 6. Percent of the mercury in blood present in the plasma I h after the intravenous administration of a 0.5 pmohkg nontoxic dose of mercuric chloride (containing 203HgC12), in rats pretreated intravenously with normal saline (0.9% aqueous NaCI) or a IO mmolkg dose of p-aminohippurate (PAH) 5.30, 60 or 180 mitt prior to the injection of HgClt. Values represent mean * S.E. for five animals per group, except for the groups of rats pretreated with saline 5 or 30 min prior to, or the group of rats pretreated with PAH 5 mitt prior to, the injection of the nontoxic dose of HgCls, in which there were four rats per group. *Significantly different (P < 0.05) from the mean for the corresponding group of control rats pretreated with normal saline. 5 min. 30 min. 60 min. 180 min. Pretreatment Time Prior To Administration of Hg

9 Saline (n = 5) 21.1 f f f f 0.60 PAH f (n=5) 23.2 * 1.02* 9.07 f zk f 1.60* 2.51 f 0.17, 5.38 f 0.71 (10 mmohkg) Values are mean f SE. Both saline and PAH were administered into the right femoral vein (4.0 ml/kg). At the end a 5-mitt pretreatment period a 0.5 mot/kg dose (in 2.0 ml/kg) of mercuric chloride (containing 203HgClr) was administered into the left femoral vein. Tissues were obtained from all the animals 24 h after the intravenous dose of mercuric chloride was administered. The total volume of blood in each animal was estimated to be 6% of weight of the animal. *Statistically different (p < 0.05) from the corresponding mean for the rats pretreated with saline. Table 3 24-h Disposition of injected inorganic mercury in rats pretreated with paminohippurate or saline Type of pretreatment Time after Animal body [Hgl in left Content of Content of Hg % Hg in blood Urinary excre- Fecal excretion pretreatment weight (g) kidney Hg in liver in blood (% present in plas- tion of Hg (% of Hg (% when Hg was ( Xi dose/g) (% dose) dose) ma dosei h) dose/24 h) injected (min)

10 32 RX. Zalups. D. W. Barfuss / Toxicology 103 (1995) COllt?X OSOM ISOM Inner Medulla Fig. 7. Content of mercury ( A dose) in the left kidney and total renal mass and concentration of mercury (% dose/g tissue) in the renal cortex, outer stripe of the outer medulla (OSOM), inner stripe of the outer medulla (ISOM) and inner medulla, 24 h after the intravenous administration of a 0.5 pmol/kg nontoxic dose of mercuric chloride (containing 203HgCl&, in rats pretreated intravenously with normal saline (0.9% aqueous NaCI) or a IO mmolikg dose of paminohippurate (PAH) 5 ruin prior to the injection of HgC12 Values represent mean f SE. for five animals per group. *Significantly different (P < 0.05) from the mean for the control group of rats pretreated with normal saline. in the group of rats pretreated with the 10 mmol/kg dose of PAH for 5 min than in the corresponding group of rats pretreated with normal saline for 5 min Intrarenal distribution of mercury. Of the zones of the kidney evaluated, the renal cortex was the only zone in which the concentration of mercury was found to be significantly different, between the group of rats pretreated with the 10 mmoykg dose of PAH and the corresponding group of rats pretreated with saline, 24 h after the intravenous injection of the 0.5 pmol/kg dose of HgC12 (Fig. 7). The concentration of mercury in the renal cortex was significantly less in the rats pretreated with PAH than in the rats pretreated with saline Disposition of mercury in blood and liver. There were no significant differences in the content of mercury in the blood or liver (24 h after the injection of the nontoxic dose of HgC12) between PAH-pretreated and saline-pretreated rats in experiment 3 (Table 3). The distribution of mercury in blood, however, was significantly different between the rats pretreated with PAH and rats pretreated with saline (Table 3). Significantly more mercury was in the plasma fraction of blood in the rats pretreated with the 10 mmol/kg dose of PAH than in the rats pretreated with saline Excretion of mercury. In this experiment, the rats pretreated with the 10 mmol/kg dose of PAH for 5 min excreted slightly, but significantly, more mercury in the urine per g kidney in 24 h than the rats pretreated with saline for 5 min (Table 3). Fecal excretion of mercury was not significantly different between the two groups of rats in experiment 3 (Table 3). 4. Discussion In the present study, the renal uptake (and/or accumulation) of intravenously administered inorganic mercury in rats was inhibited significantly in a dose-dependent and time-dependent manner by intravenous pretreatment with the water soluble organic anion p-aminohippurate (PAH). Near maximal reduction or inhibition in the renal uptake of mercury was achieved with a 10 mmol/kg dose of PAH, when it was administered 5 min prior to the intravenous injection of a 0.5 pmol/kg nontoxic dose of HgC12. However, when the dose of inorganic mercury was administered 3 h after pretreatment with a 10 mmol/kg dose of PAH, no inhibitory effect on the renal uptake of mercury could be detected, presumably because most of the administered PAH had been cleared from the blood by the kidneys. Moreover, the inhibition of the early renal uptake of mercury (induced by PAH) caused a significant reduction in the renal burden of mercury throughout the initial 24 h

11 RX. Zolups, D. W. Toxicology 103 (1995) following the injection of the HgC&. Thus, these findings support the tested hypothesis that pretreatment with an organic anion does have a significant inhibitory effect on the renal uptake (and/or accumulation) of injected inorganic mercury. Additional findings from the present study show that the reduction in the renal uptake of mercury (induced by pretreatment with PAH) is due to a reduction in the uptake of mercury in the renal cortex and in the outer stripe of the outer medulla, with a slightly greater degree of reduction occurring in the uptake of mercury in the renal cortex. In fact, in the time-dependency experiments, the renal cortical uptake (and/or accumulation) of mercury, 1 h after the administration of the 0.5 rmol/kg dose of HgCl*, was reduced by more than 40% in rats pretreated with the 10 mmol/kg dose of PAH 5 min prior to the administration of HgC12. Inhibition of thie renal uptake and/or accumulation of methylmercury has also be demonstrated after pretreatment with another well-studied organic anion. Recent findings indicate that the renal uptake and/or accumulation (Tanaka et al., 1992) and toxicity (Ban and de Ceaurriz, 1988) of methylmercury in mice are reduced significantly following pretreatment with probenecid. As mentioned previously, both PAH and probenecid are well-established competitive inhibitors of the renal transport of various organic anions (Roth-Ramel et al., 1992). Since the renal transport of organic anions (Roth-Ramel et al., 1992), and the renal uptake and accumulation of mercury (Rodier et al., 1988; Zalups and Barfuss, 1990; Zalups, 1991a,b) occur almost exclusively along segments of the proximal tubule, the collective findings from the present and previous studies tend to indicate that organic anion transport system is involved mechanistically in the renal tubular uptake of both inorganic and organic forms of mercury. It is not clear at present whether the inhibition in the uptake of mercury, induced by PAH or probenecid, occurs at the luminal and/or basolateral membrane. However, basolateral uptake would seem tlo be a logical choice based on the fact that the transport of organic anions generally occurs in a secretory direction (Roch- Ramel et al., 1992). Although the findings from this and other studies implicate the organic anion transport system as a mechanism in the renal (proximal) tubular uptake of inorganic and organic forms of mercury, this mechanism does not appear to be the only one involved in this process. This notion is supported by the fact that a substantial amount of mercury was found in the total renal mass after a period (5 min) during which near maximal inhibition of the uptake of mercury was induced with PAH. Moreover, there is other experimental evidence from rats and mice indicating that a substantial fraction of the inorganic mercury and methylmercury that is taken up by the kidney is done so by a reabsorptive mechanism involving the actions of the y-glutamyltranspeptidase (Berndt et al., 1985; Tanaka et al., 1990, 1992; Tanaka-Kagawa et al., 1993; de Ceaurriz et al., 1994; Zalups, 1994). Recent data from my laboratory indicate that this mechanism is different from the one involving the organic anion transport system (Zalups, 1994). Thus, at least two mechanisms appear to be implicated in the renal tubular uptake of inorganic (and perhaps organic) mercury. Specific details regarding the reabsorptive mechanism involving the actions of the y-glutamyltranspeptidase are lacking, although one likely possibility is that mercuric ions that are in complexes with glutathione (GSH) are taken-up across the luminal membrane of proximal tubular epithelial cells as S-conjugates of cysteinylglycine or cysteine in a sodium-dependent manner, subsequent to the enzymatic breakdown of peptide bonds in GSH. The formation of GSH S-conjugates with mercuric ions may occur directly in the plasma, whiih contains GSH at a concentration of approximately 10 PM (Lash and Jones, 1985a), or it may occur in the liver. This later notion is supported by data obtained from mice, in which the uptake, accumulation and toxicity of inorganic mercury in the kidneys were reduced significantly when hepatic GSH was depleted with 1,2-dichloro4nitrobenzene prior to the administration of inorganic mercury (Tanaka et al., 1990). Mercuric ions may also form S-conjugates with cysteine in plasma, inasmuch as the concentration of this amino acid is also around 10 pm in this compartment of blood. These conjugates may be reabsorbed directly

12 34 R. K. Zalups, D. W. Barjiis / Toxicology IO3 (1995) without requiring the actions of the y-glutamyltranspeptidase. Formation of GSH S-conjugates with mercuric ions may also be important for the entry of mercuric ions into proximal tubular epithelial cells through the mechanism involving the organic anion transport system. In vitro evidence obtained with isolated vesicles of basolateral membrane from proximal tubules indicates that GSH (as an intact tripeptide) and organic S-conjugates of GSH, such as S-( 1,2-dichlorovinyl)glutathione, can be transported across the basolateral membrane into proximal tubular epithelial cells in a sodium-dependent manner (Lash and Jones, 1983, 1984, 1985b). Additional evidence indicates that this transport process can be inhibited by the actions of the organic anion probenecid. On the basis of these findings, it is possible that mercuric ions may gain entry into proximal tubular epithelial cells by a mechanism that involves the transport of mercuric S-conjugates of GSH by the organic anion transport system. Mercuric ions may also be transported across the basolateral membrane of proximal tubular epithelial cells as some S-conjugate of cysteine. In vitro findings indicate that some S-conjugates of cysteine such as S-( 1,2-dichlorovinyl)-L-cysteine can be transported across the basolateral membrane of proximal tubular epithelial cells by a sodium-dependent and probenecid- and PAH-sensitive organic anion transport system (Lash and Anders, 1989). The notion of basolateral uptake of inorganic mercury by the organic anion transport system is further supported in part by the urinary data of the present study. At the onset of the study, it was assumed that if the PAH-sensitive mechanism for the proximal tubular uptake of mercury was present on the luminal membrane, then the level of the urinary excretion of mercury would be quite substantial. Yet, this was not the case. Even though the urinary excretion of mercury was elevated to a level that was statistically significant after pretreatment with PAH, the magnitude of the increase could not account for the large amount of mercury that was not taken up by the kidneys. This is in contrast to what occurs when the ltinal mechanism involving the y-glutamyltranspeptid- ase is inhibited with acivicin. Inhibition of the r- glutamyltranspeptidase by acivicin prior to the administration of inorganic mercury has been shown, in my laboratory (unpublished data) and in the laboratories of others (Bemdt et al., 1985; Tanaka et al., 1990), to cause a marked increase in the urinary excretion of mercury and GSH. In fact, data obtained from my laboratory indicate that the magnitude of the urinary excretion of mercury can account for the mercury that is not taken-up by the kidney. As a final point, pretreatment with PAH also caused a redistribution of mercury in the blood, without affecting the absolute amount of mercury in blood, in all experiments of the present study. More mercury was found in the plasma fraction of blood than in the cellular fraction of blood when rats were pretreated with PAH. These data tend to indicate that some uptake of inorganic mercury by erythrocytes is accomplished by the actions of an organic anion transport system that is sensitive to PAH. In conclusion, the findings from the present study indicate that the renal uptake (and/or accumulation) of intravenously administered inorganic mercury in rats can be inhibited significantly in a dose-dependent and time-dependent manner by intravenous pretreatment with the water soluble organic anion p-aminohippurate (PAH). Acknowledgements This work was supported by Grants ES05157 (RKZ) and ES05980 (DWB and RKZ) from the National Institutes of Health. References Ban, M. and de Ceaurriz, J. (1988) Probenecid-induced protection against acute hexachloro-1,3-butadiene and methyl mercury toxicity to the mouse kidney. Toxicol. Lett. 40, Berndt, W.O., Baggett, J. McC., Blacker, A. and Houser, M. (1985) Renal glutathione and mercury uptake by kidney. Fundam. Appl. Toxicol. 5, de Ceaurriz, J., Payan, J.P., Morel, G. and Brondeau, M.T. (1994) Role of extracellular glutathione and ~glutamyltranspeptidase in the disposition and kidney toxicity of inorganic mercury in rats. J. Appl. Toxicol. 24, Lash, L.H. and Jones, D.P. (1983) Transport of glutathione by

13 R. K. Zolups. D. W. / Toxitdogy 103 (1995) renal basal-lateral membrane vesicles. B&hem. Biophys. Res. Commun. 112, S-60. Lash, L.H. and Jones, D.P. (1984) Renal glutathione transport: Characteristics of the sodiumdependent system in the basallateral membrane. 1. Biol. Chem. 259, Lash, L.H. and Jones, D.P. (1985a) Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma. Arch. Biochrm. Bialphys. 240, Lash, L.H. and Jones, D.P. (1985b) Uptake of the glutathione conjugate S-(1,Zdicblorovinyl)glutathione by renal basallateral membrane vesicles and isolated kidney cells. Mol. Pharmacol. 28, Lash, L.H. and Anders, M.W. (1989) Uptake of nephrotoxic S- conjugates by isolalted rat renal proximal tubular cells. J. Pharmacol. Exp. Therap. 248, Roth-ramel, F., Besseghir, K. and Murer, H. (1992) Renal excretion and tubular itransport of organic anions and cations. In: E.E. Windhager (Ed), Handbook of Physiology, Vol. II, Section 8, Renal Physiology, Oxford University Press, New York, pp Rodier, P.M., Kates, El. and Simons, R. (1988) Mercury localization in mouse kidney over time: Autoradiography versus silver staining. Toxicol. Appl. Pharmacol. 257, Tanaka, T., Nagamuna, A. and Imura, N. (1990) Role of y- glutamyltranspeptidase in renal uptake and toxicity of inorganic mercury. Toxicology 60, Tanaka, T., Naganuma, A. and Imura, N. (1992) Routes for renal transport of methylmercury in mice. Eur. J. Pharmacol. 228, Tanaka-Kagawa, T., Naganuma, A. and Imura, N. (1993) Tubular secretion and reabsorption of mercury compounds in mouse kidney. J. Pharmacol. Exp. Therap. 264, Zalups, R.K. (1991a) Autometallographic localization of inorganic mercury in the kidneys of rats: Effect of unilateral nephrectomy and compensatory renal growth. Exp. Mol. Pathol. 54, Zalups, R.K. (1991b) Method for studying the in vivo accumulation of inorganic mercury in segments of the nephron in the kidneys of rats treated with mercuric chloride. J. Pharmacol. Methods 26, Zalups, R.K. (1995) Organic anion transport and action of y- glutamyltranspeptidase in kidney linked mechanistically to renal tubular uptake of inorganic mercury. Toxicol. Appl. Pharmacol. 132, Zalups, R.K. and Barfuss, D.W. (1990) Accumulation of inorganic mercury along the renal proximal tubule of the rabbit. Toxicol. Appl. Pharmacol. 106,