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Further study of effects of chelating agents on excretion of inorganic mercury in rats
Toxicology, 77 (1993) 157-169 Elsevier Scientific Publishers Ireland Ltd.
157
Further study of effects of chelating agents on excretion of inorganic mercury in rats Hideaki Shimada a, Satoko F u k u d o m e a, Morio K i y o z u m i b, Takayuki F u n a k o s h i a, Tatsumi Adachi c, Akira Yasutake c and Shoji Kojima a aDepartment of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862, l'Department of School Health, Faculty of Education, Kumamoto University, 40-1 Kurokami-2-chome, Kumamoto 860 and CBiochemistry Section, Department of Basic Medical Sciences, National Institute for Minamata Disease, Minamata City, Kumamoto 867 (Japan) (Received July 31st, 1992 accepted September 29th, 1992)
Summary The effects of three chelating agents, N-benzyi-D-glucamine dithiocarbamate (BGD), 2,3dimercaptopropanol (BAL) and D-penicillamine (D-PEN), on the excretion of mercury in rats exposed to mercuric chloride (HgCI2), the chemical forms of mercury compounds excreted in the bile and urine and the intestinal reabsorption of mercury compounds in the bile were studied. Rats were injected intraperitoneally with 2°3HGC12(300/~g Hg and 74 kBq of 2°DHg/kg) and 24 h later, they were injected intraperitoneally with a chelating agent (a quarter of an LDs0). The injection of the chelating agents significantly enhanced the biliary and urinary excretions of mercury. The enhancing" effect of BGD on the excretions of mercury was almost the same as that of BAL and much larger than that of D-PEN. The major chemical form of mercury in the bile and urine of rats injected with BGD after HgCI2 treatment was Hg-BGD compounds. The chemical form of mercury in the bile and urine of rats injected with BAL after HgCi2 treatment was mainly Hg-GSH compound. The mercury after HgCI2 and D-PEN treatment was excreted mainly via the urine in the form of Hg-D-PEN compound. The intestinal reabsorption of mercury from the bile of rats injected with BGD or D-PEN was only 0.18% or 0:38% of the dose, respectively. The intestinal reabsorption of mercury from the bile of rats injected with BAL was 27.38% of the dose. It was suggested that the Hg-GSH compound excreted in the bile after HgC12 and BAL treatment is partly degraded to Hg-cysteine (Cys) by the intestinal membranous enzymes and that the ligand of HgCys is replaced by BAL in the bile, resulting in the effective reabsorption of Hg-BAL compound from the intestine.
Key words: Chemical form; Inorganic mercury; N-Benzyl-D-glucamine dithiocarbamate; D-Penicillamine; 2,3-Dimercaptopropanol; Biliary and urinary excretion; Intestinal reabsorption
Introduction Mercury has been recognized as a metal highly toxic to man for many years. Inorganic mercury is predominantly accumulated in the kidney and affects the morCorrespondence to: Shoji Kojima, Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-10e-honmachi, Kurnamoto 862, Japan. 0300-483X/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
158
phology and function of the proximal tubles [1-3]. Therefore, it is important to develop an effective chelator therapy for removal of mercury to prevent mercuryinduced diseases. No satisfactory method for enhancing mercury mobilization from deposits in organs has yet been found. The most potent mercury-detoxifying agents are SH-containing compounds, of which D-penicillamine (D-PEN) and 2,3dimercaptopropanol (BAL) are used in clinical medicine. Some studies showed, however, that the injection of BAL to mice pretreated with mercury caused the redistribution of the mercury to brain [4-6]. Gabard reported that BAL caused the redistribution of mercury to the kidney in rats [7]. A number of chelating agents, such as N-acetyl-DL-penicillamine [8], 2,3-dimercaptopropane-l-sulfonate [7], 2,3dimercaptosuccinic acid [8-10] and N-(2,3-dimercaptopropyl)phthalamidic acid [11,12], have been studied as antidotes to mercury. We reported that the single and repeated injections of the new dithiocarbamate, N-benzyl-D-glucamine dithiocarbamate (BGD), into rats treated with mercuric chloride (HgC12) were effective in decreasing the mercury content in the kidney without redistribution of mercury to tissues such as the brain, heart and lung and that the reducing effect of BGD on the kidney mercury content was almost the same as that of BAL and much larger than that of D-PEN [13,14]. The present study was undertaken to further evaluate the comparative effects of BGD, BAL and D-PEN on the biliary and urinary excretions of mercury in rats exposed to HgCI2 and to examine the chemical forms and intestinal reabsorption of mercury compounds excreted in the bile and urine. Materials and methods
Materials 2°3HGC12 (specific activity, 170 MBq/mg) was obtained from New England Nuclear (Boston, MA). HgCI2 was obtained from Wako Pure Chemical Ind. (Osaka). BAL and D-PEN were obtained from Nakarai Chemicals Ltd. (Kyoto). BGD was prepared by the method reported in our previous paper [15]. Sephadex G-25 and G-75 were obtained from Pharmacia Fine Chemicals (Sweden). Structures of the chelating agents used are shown in Fig. 1. All other chemicals were of reagent grade. Each authentic mercury-chelating agent compound was prepared by mixing 0.3 mM of HgCl2 and 3.0 mM of chelating agent in 0.1 M ammonium formate buffer (pH 7.4) for 45 min at room temperature (20-25°C). Each of these preparations gave a single peak different from that of HgCl 2 on Sephadex G-25 and G-75. In situ rat biliary and urinary excretion experiment Male Wistar rats, weighing 190-220 g, were injected intraperitoneally with 2°3HGC12 (300 #g Hg and 74 kBq of 2°3Hg/kg) in a volume of 1.0 ml physiological saline and housed in individual metabolic cages with drinking water and diet (Nosan Lab Chow) ad libitum. Twenty-four hours later, the rats were anesthetized with urethane (1 g/kg intraperitoneally) and the bile duct was cannulated with polyethylene tubing (PE 10) as described previously [16]. The rats were injected intraperitoneally with BGD (2.8 mmol/kg in 2.0 ml of saline), BAL (0.213 mmol/kg in 0.1 ml of propyleneglycol), or D-PEN (0.625 mmol/kg in 0.5 ml of saline) at the
159
NaS \ C - N / C H 2 ( C H O H ) 4 C H 2 O H
S' ~
\CH 2 -
~
BGD
CH 2 -
CH -
CH2-- OH
I SH I SH BAL
C H 3 \ c -- CH-- COOH
CHfsHI
I
D-PEN
Fig. 1. Structures of chelating agents used.
dose of a quarter of an LDs0. Bile and urine samples were collected for an experimental period of 5 h. The urine in the bladder was collected by an injection syringe and combined with the urine excreted. The levels of 2°3Hg radioactivity in bile and urine were determined by using an Aloka auto well gamma scintillation counter (model ARC 300).
Collection of bile and urine samples for gel and papar chromatography Rats were injected intraperitoneally with 2°3HGC12 (300 #g Hg and 74 kBq of 2°3Hg/kg) and 24 h later, they were anesthetized with urethane and the bile duct was cannulated with polyethylene tubing. The rats were injected intraperitoneally with a chelating agent (a quarter of an LDs0) and bile samples were collected in icechilled test tubes for 1 h. Urine samples were collected from each rat for 5 h and the urine in the bladder was collected by an injection syringe and combined with the urine excreted. The levels of 2o3 Hg radioactivity in bile and urine were determined by using the gamma counter. The bile and urine samples obtained were subjected to gel and paper chromatography experiments. Gel filtration of bile and urine One milliliter of the bile or urine was applied to a Sephadex G-75 column (1.5 × 16 cm) for BGD or G-25 column (1.5 × 39 cm) for BAL and D-PEN. The column was eluted with 0.1 M ammonium formate buffer (pH 7.4) at a flow rate of 14 ml/h at room temperature and the effluent was collected in 2-ml fractions. The radioactivity of 2°3Hg in each fraction was determined by the gamma counter.
160
Paper chromatography experiment The bile or urine sample (50/xl) was subjected to paper chromatography (Advantec No. 50, Toyo Roshi) (2 x 20 cm), and the filter paper was cut in 1 cm sections. The radioactivity of 2°3Hg in each section was determined and a histogram was plotted to locate the mercury peaks on the paper.
Intestinal reabsorption experiment Rats were anesthetized with urethane and the gastrointestinal tract was exposed by a midline abdominal incision. The bile duct was cannulated with polyethylene tubing. Bile samples containing mercury (Hg and 2°3Hg) were introduced into the duodenum by a syringe. Bile and urine samples were collected from each rat for 24 h and were counted in the gamma counter for 2°3Hg radioactivity. The rats were killed by decapitation at the end of the experiment and the liver, kidney, and carcass (residue after removal of liver, kidney and gastrointestinal tract including contents from the whole body) were removed for 2°3Hg counting in the gamma counter. The percentages of mercury absorbed were calculated by the following equation: 2°3Hg radioactivity in bile, urine, liver, kidney and carcass x 100
Percentage absorbed = 2°3Hg radioactivity in sample administered
Determination of BAL in bile Rats were anesthetized with urethane and the bile duct was cannulated with polyethylene tubing. Bile samples were collected in ice-chilled test tubes for 1 h after administration of BAL (0.213 mmol/kg). The BAL in the bile samples was determined by high performance liquid chromatography (HPLC) using ammonium 7fluorobenzo-2-oxa-l,3-diazole-4-sulfonate (SBD-F) as fluorogenic reagent according to the method of Toyooka and Imai [17].
Statistical analysis Data were analyzed by one-way analysis of variance. When the analysis indicated a significant difference, the treated groups were compared to the controls by Duncan's new multiple range test. R ~
Table I shows the biliary and urinary excretions of mercury when each chelating agent was injected 24 h after HgC12 treatment. BGD and BAL significantly enhanced both the biliary and urinary excretions of mercury to a large extent, while D-PEN significantly enhanced the urinary excretion of the metal. The cumulative biliary and urinary excretions of mercury in a 5 h period after treatment with BGD or BAL was significantly larger than that with D-PEN. The characterization of mercury in the urine after injection of BGD, BAL and DPEN in rats exposed to HgCI 2 24 h earlier was performed by both gel and paper chromatographic methods (Figs. 2 and 3). As shown in Figs. 2A1 and A2, the
161
TABLE I EFFECTS OF CHELATING AGENTS ON BILIARY AND URINARY EXCRETIONS OF MERCURY IN RATS PRETREATED WITH MERCURY, 24 h EARLIER Rats were injected i.p. with 2°3HGC12(300 t~g Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, they were injected i.p. with saline or chelating agents (a quarter of an LDso) and then bile and urine samples were collected for 5 h. The values represent the mean ± S.D. (n = 4). Chelating agent
Mercury excreted/5h (% of dose) Bile
Control BGD BAL D-PEN
0.52 7.86 7.32 0.88
Urine ±0.08 4- 2.15 a'b 4- 2.26 a'b 4- 0.09 a
1.73 12.48 12.76 3.75
4- 0.56 4- 1.53a'b 4- 7.82 a 4- 1.08a
aSignificantly different from control (P < 0.05). bSignificantly different from D-PEN (P < 0.05). A (Hg alone) B (Hg-BGD)
20-
Hg-BGD
'iJ
Hg-GSH
15
1
,
40~ A2 30 "t
Bd
5 0
1.0
&
30-
2'0
c (Hg-BAL)
o
3'0
Hg-GSH
~'0
2'0
3'0
D (Hg-D-PEN)
60.
Hg-D-PEN
4,
50-
~,
Bd
20-
40.
~. 10-
/~
Hg-BAL
° t2. 1'0
2'0
3'0
4'0
20
Hg-GSH
30. 20. 10'
0
1'0
2'0
30
2o
Fraction number Fig. 2. Gel chromatographic profiles of rat urine after treatment with mercury and chelating agents. Rats were injected i.p. with 2°3HGC12 (300 ~g Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, the rats were injected i.p. with BGD, BAL, or o-PEN (a quarter of an LDs0) and urine samples were collected tor 5 h. The urine samples were fractionated on Sephadex G-75 column (1.5 x 16 cm) (A1 and B) or G-25 column (1.5 x 39 cm) (A2, C and D). Bd, blue dextran. GSH, glutathione.
162 A (Hg-BGD) 10C
-
-
Hg-GSH
81360 - I Hg-BGD
4G-
B (Hg-BAL) .~
100-
Hg-GSH
80,-
~
40
ill
C (Hg-D-PEN)
80
Hg-GSH H~-D-PEN
20 0 origin
front
Fig. 3. Paper chromatography of rat urine after treatment with mercury and chelating agents. Rats were injected i.p. with 2°3HGC12(300/~g Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, the rats were injected i.p. with BGD, BAL, or D-PEN (a quarter of an LDs0) and urine samples were collected for 5 h. The urine sample was applied to filter paper. Solvent system: BGD, ethanol-0.1 M ammonium formate buffer (pH 7.4) (7:3); BAL, dioxane-0.1 M ammonium formate buffer (pH 7.4) (9:1); D-PEN, 0.1 M ammonium formate buffer (pH 7.4).
S e p h a d e x G-75 (A1) a n d G-25 (A2) c h r o m a t o g r a p h i c profiles o f the urine after a d m i n i s t r a t i o n o f HgCI2 a l o n e showed t h a t m o s t o f the excreted m e r c u r y was present in fraction 14 (G-75) a n d in f r a c t i o n 25 (G-25) c o r r e s p o n d i n g to H g - G S H c o m p o u n d , respectively. T h e S e p h a d e x G-75 c h r o m a t o g r a p h i c profile o f the urine o f rats injected with B G D after m e r c u r y t r e a t m e n t is shown in Fig. 2B. T h r e e p e a k s with
163
fractions 7, 14 and 28 corresponded to those of Hg-high molecular weight compound (HMW, probably protein), Hg-GSH and Hg-BGD compounds, respectively. The Sephadex G-25 chromatographic profile of the urine of rats injected with BAL after mercury treatment showed the main peak with fraction 25 corresponding to HgGSH compound and the small peak with fractions 14 and 36 corresponding to HgHMW and Hg-BAL compounds, respectively (Fig. 2C). The gel chromatographic profile of the urine after treatment with mercury and D-PEN showed one peak with fraction 31 corresponding to Hg-D-PEN compound (Fig. 2D). From further characterization of the mercury-binding compound in the urine by paper chromatography, the distribution pattern of 2°3Hg radioactivity detected on the chromatogram of urine after treatment with BGD, BAL, or D-PEN corresponded to that of Hg-GSH and Hg-BGD, Hg-GSH, or Hg-D-PEN compound, respectively (Fig. 3A-C). Furthermore, the characterization of mercury in the bile after treatment with HgC12 and the chelating agents was investigated by the gel chromatographic method (Fig. 4). The Sephadex G-75 (Fig. 4A1) and G-25 (Fig. 4A2) A (Hg alone) 20
20-
Bd
:tYL____ i
i
!
i
i
B (Hg-BGD) Hg-GSH Bd
,o-2j
Hg-BGD
5-
10 20'
C (Hg-BAL)
20
30
1'0
25. D (Hg-D-PEN) Bd
Hg-~SH
Hg-~SH
15"
0
40
Hg-BAL
10'
20
30
40
Hg-GSH
15.
- ~
10.
5'
5.
0
0. 10
2'0
3'0
40
0
1.0
2'0
3'0
4~
Fractionnumber Fig. 4. Gel chromatographic profiles of rat bile after treatment with mercury and chelating agents. Rats were injected i.p. with 2°3HGC12(300/~g Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, the rats were injected i.p. with BGD, BAL, or D-PEN (a quarter of an LD50) and bile samples were collected for 1 h. The bile samples we/'e fractionated on Sephadex G-75 column (I.5 x 16 era) (A1 and B) or G-25 column (1.5 x 39 cm) (A2, C and D). Bd, blue dextran. GSH, glutathione.
164
chromatographic profiles of the bile after administration of HgCI 2 alone showed two peaks with fractions 7 and 14 on G-75 and fractions 14 and 25 on G-25, respectively. In Fig. 4A1 and A2, the first small peak corresponded to Hg-HMW compound and the second main peak corresponded to Hg-GSH compound. The gel chromatographic profile of the bile after treatment with HgCI2 and BGD showed three peaks with fractions 7, 14 and 28 corresponding to Hg-HMW, Hg-GSH and A (Hg-BGD) 10(]
Hg-GSH
8~3 6G Hg-BGD 4~ 2G
B (Hg-BAL) IO0o)
.~
Hg-GSH
80-
g 60 ~o
g-
40
o
lF
C (Hg-D-PEN)
8O
Hg-GSH H~-D-PEN
20 0 origin
front
Fig. 5. Paper chromatography of rat bile after treatment with mercury and chelating agents. Rats were injected i.p. with 2°3HGC12(300 #g Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, the rats were injected i.p. with BGD, BAL, or D-PEN (a quarter of an LDs0 ) and bile samples were collected for 1 h. The bile sample was applied to filter paper. Solvent system: BGD, ethanol-0.1 M ammonium formate buffer (pH 7.4) (7:3); BAL, dioxane-0.1 M ammonium formate buffer (pH 7.4) (9:1); D-PEN, 0.1 M ammonium formate buffer (pH 7.4).
165 H g - B G D compounds, respectively (Fig. 4B). The diagram of the bile after administration of HgCI2 and BAL showed three peaks with fractions 14, 25 and 36 corresponding to H g - H M W , H g - G S H and Hg-BAL compounds (Fig. 4C). The gel chromatographic profile of bile after treatment with HgCI2 and D-PEN showed three peaks corresponding to H g - H M W compound (fraction 14), unknown Hg compound (fraction 22) and H g - G S H compound (fraction 25), respectively (Fig. 4D). The paper chromatographic histograms of the bile of rats treated with the chelating agents after mercury injection are shown in Fig. 5. The diagram of bile after administration of HgC12 and B G D showed two mercury compounds corresponding to H g - G S H and H g - B G D compounds (Fig. 5A). The chromatographic profiles of bile of rats injected with BAL or D-PEN after HgC12 treatment also showed the presence of Hg-GSH, respectively (Fig. 5B and C). Table II shows the percentages of mercury compounds in bile and urine of rats injected with chelating agents after HgCI2 treatment. In the bile and urine of rats treated with HgCI2 and BGD, H g - B G D compound was major and H g - G S H and H g - H M W compounds were minor. The percentages of H g - G S H and Hg-BAL compounds in the bile and urine of rats treated with HgC12 and BAL were 90.7 and 7.6% in the bile and 90.5 and 6.2% in the urine, respectively. The percentages of HgG S H and unknown Hg compounds in the bile after HgC12 and D-PEN treated were 48.4 and 39.9%, respectively, but only Hg-D-PEN compound was excreted in the urine. We further investigated the small intestinal reabsorption of mercury compounds from the bile of rats injected with the chelating agents 24 h after HgC12 treatment. TABLE II PERCENTAGES OF MERCURY COMPOUNDS IN BILE AND URINE OF RATS INJECTED WITH CHELATING AGENTS AFTER MERCURY TREATMENT Rats were injected i.p. with 2°3HGC12(300 ~g Hg and 74 kBq of 2°3Hg/kg).Twenty-fourhours later, the rats were injected i.p. with the chelating agents (a quarter of an LDs0). The bile and urine sampleswere collected for 1 and 5 h, respectivelyand fractionated on Sephadex G-75 column for BGD or Sephadex G-25 column for BAL and ~PEN. The values represent the mean ± S.D. (n = 3). Chelating agent
Mercury compound
Percentage of mercury detected Bile
BGD BAL D-PEN
Hg-BGD Hg-GSH Hg-HMW Hg-GSH Hg-BAL Hg-HMW Hg-D-PEN Hg-GSH Unknown Hg Hg-HMW
63.4 ± 27.6 ± 9.1 ± 90.7 ± 7.6 ± 1.7 ± -48.4 ± 39.6 ± 12.0 ±
Urine 11.2 8.5 2.8 1.8 2.7 0.9
82.9 + 3.1 12.3 ± 3.7 4.8 ± 3.8 90.5 ± 3.1 6.2 ± 2.5 3.3 ±2.1 100.0 ± 0.0
13.1 17.7 5.3
GSH, glutathione; HMW, high molecular weight compound.
166 TABLE III IN SITU SMALL COMPLEXES
INTESTINAL
ABSORPTION
OF
MERCURY-CHELATING
AGENTS
Rats were injected i.p. with 2°3HGC12 (300/zg Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, the rats were injected i.p. with the chelating agents (a quarter of an LDs0) and bile samples were collected for 1 h. The values represent the mean 4- S.D. (n = 3). N.D., not detected. Hg absorbed (% of dose) Hg-BGD Bile of rat treated with Hg-BGD Hg-BGD compound Hg-GSH compound Hg-BAL Bile of rat treated with Hg-BAL Hg-BAL compound Hg-BAL compound in bile Hg-BAL compound + glycocholic acid Hg-Cys compound Hg-D-PEN Bile of rat treated with Hg-D-PEN Hg-D-PEN compound
0.18 4- 0.05 N.D. N.D. 27.38 9.91 18.72 22.93 2.39
± 5.79 ± 0.61 4- 2.28 4- 4.73 4- 0.58
0.38 4- 0.37 N.D.
GSH, glutathione; Cys, cysteine.
CG GSH
BAL
O
Cy
!
I
I
I
I
I
I
0
5
10
15
20
25
30
Retention time (rain) Fig. 6. HPLC profile of rat bile after treatment with BAL. Rats were injected i.p. with BAL (0.213 mmol/kg) and bile samples were collected for 1 h. Peaks were identified as follows: Cys, cysteine; CG, cysteinylglycine; GSH, glutathione.
167
As shown in Table III, the intestinal reabsorption of mercury from the bile of rats treated with BGD or D-PEN was only 0.18% or 0.38% of the dose, respectively. However, the intestinal reabsorption of mercury from the bile of rats injected with BAL was 27.38% of the dose. Hg-GSH compound, which was characterized as the main mercury compound in the bile of rats injected with BAL, was little absorbed from the intestinal tracts. On the other hand, the intestinal absorption of Hg-BAL compound or Hg-BAL compound in bile collected from the control rat was shown to be 9.91% or 18.72% of the dose, respectively. In addition, the intestinal absorption of mercury from Hg-BAL compound was increased by the addition of glycocholic acid which is the major constituent of bile acids. As shown in Fig. 6, also, the excretion of BAL in the bile of rats injected with BAL was determined by HPLC method. Discussion
The purpose of the present study was to evaluate the effects of BGD, BAL and D-PEN on the excretion of mercury and to elucidate the chemical characterization and intestinal reabsorption of mercury compounds in the bile and urine of rats injected with the chelating agents after HgCI2 treatment. The injection of the chelating agents at 24 h after mercury treatment showed that BGD and BAL enhanced both the biliary and urinary excretions of mercury and that D-PEN enhanced mainly the uriffary excretion of the metal (Table I). The enhancing effect of BGD on the biliary and urinary excretions of mercury was almost the same as that of BAL and much larger than that o f D-PEN. From chemical characterization of the bile and urine of rats after HgC12 injection using both gel and paper chromatographic methods, the mercury was mainly bound to GSH. The major chemical form of mercury in the bile and urine of rats injected with BGD after HgC12 treatment was mainly Hg-BGD compound in both the bile and urine. This finding suggests that BGD enhances mainly the mercury excretion following HgC12 injection based on the formation of Hg-BGD compound. The chemical form of mercury in the bile and urine of rats injected with BAL after HgCI2 treatment was mainly Hg-GSH compound. It has been shown that the stability constant (log k = 41.6) of Hg-GSH compound [18] is much larger than that (log k = 25.74) of Hg-BAL compound [19]. Our results, which examined the difference in the affinity of mercury for GSH and BAL using the gel chromatographic method, also indicated that the chemical forms of mercury (Hg 2÷) in equimolar mixtures of the two thiols were Hg-GSH compound (90% of applied 2°3Hg) and Hg-BAL compound (10% of applied 2°3Hg) (data not shown). These findings suggest that the replacement of the ligands in Hg-BAL and/or Hg-BAL metabolites by GSH would occur in the body and that the mercury in the body is mainly excreted into the bile and urine in the form of Hg-GSH compound. The mercury after HgC12 and D-PEN treatment was excreted mainly via the urine in the form of Hg-D-PEN compound. In the bile of the minor excretory route of mercury after HgC12 and DPEN injection, the mercury was mainly present in the forms of Hg-GSH and unknown Hg compounds.
168
It is known that BAL effectively reduces the kidney level of inorganic mercury when used as an antidote but there is an adverse side effect in that redistribution of mercury to other organs occurs simultaneously [20,21]. We also reported that the injection of BAL at 24 h after mercury treatment caused the redistribution of mercury to the heart, lung and brain [14,22]. Therefore, we investigated the intestinal reabsorption of mercury compounds from the bile of rats injected with the chelating agents after HgC12 treatment (Table III). The intestinal reabsorption of mercury from the bile of rats injected with BGD and D-PEN was negligible, but that of rats injected with BAL was considerably large. Although the chemical form of mercury in the bile of rats injected with BAL after HgCI2 treatment was mainly Hg-GSH compound, Hg-GSH compound was little absorbed from the intestine. It is well known that GSH and its conjugates are degraded to cysteinylglycine and cysteine (Cys) by 7-glutamyl transpeptidase (7-GTP) and dipeptidase [23, 24]. The stability constant of Hg-BAL is much lower than that of Hg-GSH and much larger than that of Hg-Cys (log k = 14.21) [25]. In addition, BAL was found in the bile after injection of the chelating agent (Fig. 3). These findings indicate that Hg-GSH compound excreted in the bile is partly degrated to Hg-Cys form by the intestinal membranous "¥-GTP and dipeptidase and then Hg bound Cys is exchanged for BAL. Hg-BAL compound produced can be efficiently reabsorbed from the intestinal tract and the absorption of Hg-BAL compound is strongly stimulated by glycocholic acid which is a constituent of bile. The redistribution of mercury to tissues of rats treated with BAL after HgCI2 injection is considered to be due to the intestinal reabsorption of mercury compounds excreted in the bile. References 1
G.E. Voigt, Histochemishe Untersuchungen fiber die Verteilung des Quecksilbers bei experimenteller Sublimatvergiftung. Acta Pathol. Microbiol. Scand., 43 0958) 321. 2 F. Timm and R. Neth, Die normalen Schwermetalle der Niere. Histochemie, I (1959) 403. 3 F. Timm and M. Arnord, Der cellulare Verbleid kleiner Quecksilbermengen in der Rattenniere. Naunyn Schmiedebergs Arch. Exp. Pathol., 239 (1960) 393. 4 J. Aaseth, J. Alexand and N. Raknerud, Treatment of mercuric chloride poisoning with dimercaptosuccinic acid and diuretics: Preliminary studies. J. Toxicol.-din. Toxicol., 19 (1982) 173. 5 V. Eybl, J. Sykora, M. Koutenska, J. Koutenska and F. Mertl, Effect of spilonolactone, thiomestrone and dimercaprol on the toxicity, retention and distribution of mercury in the mouse. Arzneim.-Forsch., 23 (1973) 867. 6 A. Wannag and J. Aaseth, The effect of immediate and delayed treatment with 2,3dimercaptopropane-l-sulfonate on the distribution and toxicity of inorganic mercury in mice and in foetal and adult rats. Acta Pharmacol. Toxicol., 46 (1980) 81. 7 B. Gabard, The excretion and distribution of inorganic mercury in rats as influenced by several chelating agents. Arch. Toxicol., 35 (1976) 15. 8 H.V. Aposian, Penicillamine and its analogue chelating agents. Ann. N.Y. Acad. Sci., 179 (1971) 481. 9 M. Magos, The effects of dimercaptosuccinic acid on the excretion and distribution of mercury in rats and mice treated with mercuric chloride and methylmercuric chloride. Br. J. Pharmacol., 56 (1976) 479. 10 J. Aaseth and E. Friedheim, Treatment of methyl mercury poisoning in mice with 2,3dimercaptosuccinic acid and other complexing thiol. Acta Pharmacol. Toxicol., 42 (1978) 248. l 1 K. Morita and T. Yonaga, Effects of N-(2,3-dimercaptopropyl)phthalamidic acid on the biliary excretion of mercury compounds. Jap. J. Pharmacol., (Suppl.) 26 (1976) 85.
169 12 T. Yonaga and K. Morita, Comparison of the effect of N-(2,3-dimercaptopropyl) phthalamidic acid, DL-penicillamine, and dimercaproi on the excretion of tissue retention of mercury in mice. Toxicol. Appl. Pharmacol., 57 (1981) 197. 13 H. Shimada, M. Kiyozumi, T. Honda and S. Kojima, Studies on poisonous metals. XX. Effects of chelating agents on distribution and excretion of inorganic mercury in rats. Yakugaku Zasshi, 108 (1988) 1209. 14 S. Kojima, H. Shimada and M. Kiyozumi, Comparative effects of chelating agents on distribution, excretion and renal toxicity of inorganic mercury in rats. Res. Commun. Chem. Pathol. Pharmacol., 64 (1989) 471. 15 S. Kojima, K, Kaminaka, M. Kiyozumi and T. Honda, Comparative effects of three chelating agents on distribution and excretion of cadmium in rats. Toxicol. Appl. Pharmacol., 83 (1986) 516. 16 S. Kojima, M. Kiyozumi and K. Saito, Studies on poisonous metals. II. Effect of chelating agents on excretion of cadmium through bile and gastrointestinal mucosa in rats. Chem. Pharm. Bull., 24 (1976) 16. 17 T. Toyooka and K. Imai, High-performance liquid chromatography and fluorometric detection of biologically important thiols, derivatized with ammonium 7-fluorobenzo-2-oxa-l,3-diazole-4sulphonate (SBD-F). J. Chromatogr., 282 (1983) 495. 18 W. Stricks and I.M. Kolthoff, Detections between mercuric mercury and cysteine and glutathione. Apparent dissociation constants, heats and entropies of formation of various forms of mercuric mercapto-cysteine and -glutathione. J. Am. Chem. Soc., 75 (1953) 5673. 19 R.L. Coates and M.M. Jones, Stability constants of the mercury (2]-BAL complexes. J. Inorg. Nucl. Chem., 39 (1977) 677. 20 M. Berlin and T. Lewander, Increased brain uptake of Hg caused by 2,3-dimercap-topropanol in mice given HgCl2. Acta Pharmacol. Toxicol., 22 (1965) 1. 21 L. Magos, Effect of 2,3-dimercaptopropanol (BAL) on urinary excertion and brain content of mercury. Arch. Pharmacol., 259 (1968) 183. 22 M. Kiyozumi, H. Shimada, T. Honda and S. Kojima, Studies on poisonous metals. XIX. Comparative effects of chelating agents on distribution and excretion of inorganic mercury in rats. Chem. Pharm. Bull., 36 (1988) 2599. 23 A. Meister and S.S. Tate, Giutathione and related ).-glutamyl compounds: biosynthesis and utilization. Annu. Rev. Biochem., 45 (1976) 559. 24 T. McIntyre and N.P. Curthoys, Renal catabolism of glutathione: characterization of a particulate rat renal dipeptidase that catalyzes the hydrolysis of cysteinylglycine. J. Biol. Chem., 257 (1982) 11915. 25 G.R. Lenz and A.E. Martell, Metal chelates of some sulfur-containing amino acids. Biochemistry, 3 (1964) 745.
157
Further study of effects of chelating agents on excretion of inorganic mercury in rats Hideaki Shimada a, Satoko F u k u d o m e a, Morio K i y o z u m i b, Takayuki F u n a k o s h i a, Tatsumi Adachi c, Akira Yasutake c and Shoji Kojima a aDepartment of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862, l'Department of School Health, Faculty of Education, Kumamoto University, 40-1 Kurokami-2-chome, Kumamoto 860 and CBiochemistry Section, Department of Basic Medical Sciences, National Institute for Minamata Disease, Minamata City, Kumamoto 867 (Japan) (Received July 31st, 1992 accepted September 29th, 1992)
Summary The effects of three chelating agents, N-benzyi-D-glucamine dithiocarbamate (BGD), 2,3dimercaptopropanol (BAL) and D-penicillamine (D-PEN), on the excretion of mercury in rats exposed to mercuric chloride (HgCI2), the chemical forms of mercury compounds excreted in the bile and urine and the intestinal reabsorption of mercury compounds in the bile were studied. Rats were injected intraperitoneally with 2°3HGC12(300/~g Hg and 74 kBq of 2°DHg/kg) and 24 h later, they were injected intraperitoneally with a chelating agent (a quarter of an LDs0). The injection of the chelating agents significantly enhanced the biliary and urinary excretions of mercury. The enhancing" effect of BGD on the excretions of mercury was almost the same as that of BAL and much larger than that of D-PEN. The major chemical form of mercury in the bile and urine of rats injected with BGD after HgCI2 treatment was Hg-BGD compounds. The chemical form of mercury in the bile and urine of rats injected with BAL after HgCi2 treatment was mainly Hg-GSH compound. The mercury after HgCI2 and D-PEN treatment was excreted mainly via the urine in the form of Hg-D-PEN compound. The intestinal reabsorption of mercury from the bile of rats injected with BGD or D-PEN was only 0.18% or 0:38% of the dose, respectively. The intestinal reabsorption of mercury from the bile of rats injected with BAL was 27.38% of the dose. It was suggested that the Hg-GSH compound excreted in the bile after HgC12 and BAL treatment is partly degraded to Hg-cysteine (Cys) by the intestinal membranous enzymes and that the ligand of HgCys is replaced by BAL in the bile, resulting in the effective reabsorption of Hg-BAL compound from the intestine.
Key words: Chemical form; Inorganic mercury; N-Benzyl-D-glucamine dithiocarbamate; D-Penicillamine; 2,3-Dimercaptopropanol; Biliary and urinary excretion; Intestinal reabsorption
Introduction Mercury has been recognized as a metal highly toxic to man for many years. Inorganic mercury is predominantly accumulated in the kidney and affects the morCorrespondence to: Shoji Kojima, Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-10e-honmachi, Kurnamoto 862, Japan. 0300-483X/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
158
phology and function of the proximal tubles [1-3]. Therefore, it is important to develop an effective chelator therapy for removal of mercury to prevent mercuryinduced diseases. No satisfactory method for enhancing mercury mobilization from deposits in organs has yet been found. The most potent mercury-detoxifying agents are SH-containing compounds, of which D-penicillamine (D-PEN) and 2,3dimercaptopropanol (BAL) are used in clinical medicine. Some studies showed, however, that the injection of BAL to mice pretreated with mercury caused the redistribution of the mercury to brain [4-6]. Gabard reported that BAL caused the redistribution of mercury to the kidney in rats [7]. A number of chelating agents, such as N-acetyl-DL-penicillamine [8], 2,3-dimercaptopropane-l-sulfonate [7], 2,3dimercaptosuccinic acid [8-10] and N-(2,3-dimercaptopropyl)phthalamidic acid [11,12], have been studied as antidotes to mercury. We reported that the single and repeated injections of the new dithiocarbamate, N-benzyl-D-glucamine dithiocarbamate (BGD), into rats treated with mercuric chloride (HgC12) were effective in decreasing the mercury content in the kidney without redistribution of mercury to tissues such as the brain, heart and lung and that the reducing effect of BGD on the kidney mercury content was almost the same as that of BAL and much larger than that of D-PEN [13,14]. The present study was undertaken to further evaluate the comparative effects of BGD, BAL and D-PEN on the biliary and urinary excretions of mercury in rats exposed to HgCI2 and to examine the chemical forms and intestinal reabsorption of mercury compounds excreted in the bile and urine. Materials and methods
Materials 2°3HGC12 (specific activity, 170 MBq/mg) was obtained from New England Nuclear (Boston, MA). HgCI2 was obtained from Wako Pure Chemical Ind. (Osaka). BAL and D-PEN were obtained from Nakarai Chemicals Ltd. (Kyoto). BGD was prepared by the method reported in our previous paper [15]. Sephadex G-25 and G-75 were obtained from Pharmacia Fine Chemicals (Sweden). Structures of the chelating agents used are shown in Fig. 1. All other chemicals were of reagent grade. Each authentic mercury-chelating agent compound was prepared by mixing 0.3 mM of HgCl2 and 3.0 mM of chelating agent in 0.1 M ammonium formate buffer (pH 7.4) for 45 min at room temperature (20-25°C). Each of these preparations gave a single peak different from that of HgCl 2 on Sephadex G-25 and G-75. In situ rat biliary and urinary excretion experiment Male Wistar rats, weighing 190-220 g, were injected intraperitoneally with 2°3HGC12 (300 #g Hg and 74 kBq of 2°3Hg/kg) in a volume of 1.0 ml physiological saline and housed in individual metabolic cages with drinking water and diet (Nosan Lab Chow) ad libitum. Twenty-four hours later, the rats were anesthetized with urethane (1 g/kg intraperitoneally) and the bile duct was cannulated with polyethylene tubing (PE 10) as described previously [16]. The rats were injected intraperitoneally with BGD (2.8 mmol/kg in 2.0 ml of saline), BAL (0.213 mmol/kg in 0.1 ml of propyleneglycol), or D-PEN (0.625 mmol/kg in 0.5 ml of saline) at the
159
NaS \ C - N / C H 2 ( C H O H ) 4 C H 2 O H
S' ~
\CH 2 -
~
BGD
CH 2 -
CH -
CH2-- OH
I SH I SH BAL
C H 3 \ c -- CH-- COOH
CHfsHI
I
D-PEN
Fig. 1. Structures of chelating agents used.
dose of a quarter of an LDs0. Bile and urine samples were collected for an experimental period of 5 h. The urine in the bladder was collected by an injection syringe and combined with the urine excreted. The levels of 2°3Hg radioactivity in bile and urine were determined by using an Aloka auto well gamma scintillation counter (model ARC 300).
Collection of bile and urine samples for gel and papar chromatography Rats were injected intraperitoneally with 2°3HGC12 (300 #g Hg and 74 kBq of 2°3Hg/kg) and 24 h later, they were anesthetized with urethane and the bile duct was cannulated with polyethylene tubing. The rats were injected intraperitoneally with a chelating agent (a quarter of an LDs0) and bile samples were collected in icechilled test tubes for 1 h. Urine samples were collected from each rat for 5 h and the urine in the bladder was collected by an injection syringe and combined with the urine excreted. The levels of 2o3 Hg radioactivity in bile and urine were determined by using the gamma counter. The bile and urine samples obtained were subjected to gel and paper chromatography experiments. Gel filtration of bile and urine One milliliter of the bile or urine was applied to a Sephadex G-75 column (1.5 × 16 cm) for BGD or G-25 column (1.5 × 39 cm) for BAL and D-PEN. The column was eluted with 0.1 M ammonium formate buffer (pH 7.4) at a flow rate of 14 ml/h at room temperature and the effluent was collected in 2-ml fractions. The radioactivity of 2°3Hg in each fraction was determined by the gamma counter.
160
Paper chromatography experiment The bile or urine sample (50/xl) was subjected to paper chromatography (Advantec No. 50, Toyo Roshi) (2 x 20 cm), and the filter paper was cut in 1 cm sections. The radioactivity of 2°3Hg in each section was determined and a histogram was plotted to locate the mercury peaks on the paper.
Intestinal reabsorption experiment Rats were anesthetized with urethane and the gastrointestinal tract was exposed by a midline abdominal incision. The bile duct was cannulated with polyethylene tubing. Bile samples containing mercury (Hg and 2°3Hg) were introduced into the duodenum by a syringe. Bile and urine samples were collected from each rat for 24 h and were counted in the gamma counter for 2°3Hg radioactivity. The rats were killed by decapitation at the end of the experiment and the liver, kidney, and carcass (residue after removal of liver, kidney and gastrointestinal tract including contents from the whole body) were removed for 2°3Hg counting in the gamma counter. The percentages of mercury absorbed were calculated by the following equation: 2°3Hg radioactivity in bile, urine, liver, kidney and carcass x 100
Percentage absorbed = 2°3Hg radioactivity in sample administered
Determination of BAL in bile Rats were anesthetized with urethane and the bile duct was cannulated with polyethylene tubing. Bile samples were collected in ice-chilled test tubes for 1 h after administration of BAL (0.213 mmol/kg). The BAL in the bile samples was determined by high performance liquid chromatography (HPLC) using ammonium 7fluorobenzo-2-oxa-l,3-diazole-4-sulfonate (SBD-F) as fluorogenic reagent according to the method of Toyooka and Imai [17].
Statistical analysis Data were analyzed by one-way analysis of variance. When the analysis indicated a significant difference, the treated groups were compared to the controls by Duncan's new multiple range test. R ~
Table I shows the biliary and urinary excretions of mercury when each chelating agent was injected 24 h after HgC12 treatment. BGD and BAL significantly enhanced both the biliary and urinary excretions of mercury to a large extent, while D-PEN significantly enhanced the urinary excretion of the metal. The cumulative biliary and urinary excretions of mercury in a 5 h period after treatment with BGD or BAL was significantly larger than that with D-PEN. The characterization of mercury in the urine after injection of BGD, BAL and DPEN in rats exposed to HgCI 2 24 h earlier was performed by both gel and paper chromatographic methods (Figs. 2 and 3). As shown in Figs. 2A1 and A2, the
161
TABLE I EFFECTS OF CHELATING AGENTS ON BILIARY AND URINARY EXCRETIONS OF MERCURY IN RATS PRETREATED WITH MERCURY, 24 h EARLIER Rats were injected i.p. with 2°3HGC12(300 t~g Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, they were injected i.p. with saline or chelating agents (a quarter of an LDso) and then bile and urine samples were collected for 5 h. The values represent the mean ± S.D. (n = 4). Chelating agent
Mercury excreted/5h (% of dose) Bile
Control BGD BAL D-PEN
0.52 7.86 7.32 0.88
Urine ±0.08 4- 2.15 a'b 4- 2.26 a'b 4- 0.09 a
1.73 12.48 12.76 3.75
4- 0.56 4- 1.53a'b 4- 7.82 a 4- 1.08a
aSignificantly different from control (P < 0.05). bSignificantly different from D-PEN (P < 0.05). A (Hg alone) B (Hg-BGD)
20-
Hg-BGD
'iJ
Hg-GSH
15
1
,
40~ A2 30 "t
Bd
5 0
1.0
&
30-
2'0
c (Hg-BAL)
o
3'0
Hg-GSH
~'0
2'0
3'0
D (Hg-D-PEN)
60.
Hg-D-PEN
4,
50-
~,
Bd
20-
40.
~. 10-
/~
Hg-BAL
° t2. 1'0
2'0
3'0
4'0
20
Hg-GSH
30. 20. 10'
0
1'0
2'0
30
2o
Fraction number Fig. 2. Gel chromatographic profiles of rat urine after treatment with mercury and chelating agents. Rats were injected i.p. with 2°3HGC12 (300 ~g Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, the rats were injected i.p. with BGD, BAL, or o-PEN (a quarter of an LDs0) and urine samples were collected tor 5 h. The urine samples were fractionated on Sephadex G-75 column (1.5 x 16 cm) (A1 and B) or G-25 column (1.5 x 39 cm) (A2, C and D). Bd, blue dextran. GSH, glutathione.
162 A (Hg-BGD) 10C
-
-
Hg-GSH
81360 - I Hg-BGD
4G-
B (Hg-BAL) .~
100-
Hg-GSH
80,-
~
40
ill
C (Hg-D-PEN)
80
Hg-GSH H~-D-PEN
20 0 origin
front
Fig. 3. Paper chromatography of rat urine after treatment with mercury and chelating agents. Rats were injected i.p. with 2°3HGC12(300/~g Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, the rats were injected i.p. with BGD, BAL, or D-PEN (a quarter of an LDs0) and urine samples were collected for 5 h. The urine sample was applied to filter paper. Solvent system: BGD, ethanol-0.1 M ammonium formate buffer (pH 7.4) (7:3); BAL, dioxane-0.1 M ammonium formate buffer (pH 7.4) (9:1); D-PEN, 0.1 M ammonium formate buffer (pH 7.4).
S e p h a d e x G-75 (A1) a n d G-25 (A2) c h r o m a t o g r a p h i c profiles o f the urine after a d m i n i s t r a t i o n o f HgCI2 a l o n e showed t h a t m o s t o f the excreted m e r c u r y was present in fraction 14 (G-75) a n d in f r a c t i o n 25 (G-25) c o r r e s p o n d i n g to H g - G S H c o m p o u n d , respectively. T h e S e p h a d e x G-75 c h r o m a t o g r a p h i c profile o f the urine o f rats injected with B G D after m e r c u r y t r e a t m e n t is shown in Fig. 2B. T h r e e p e a k s with
163
fractions 7, 14 and 28 corresponded to those of Hg-high molecular weight compound (HMW, probably protein), Hg-GSH and Hg-BGD compounds, respectively. The Sephadex G-25 chromatographic profile of the urine of rats injected with BAL after mercury treatment showed the main peak with fraction 25 corresponding to HgGSH compound and the small peak with fractions 14 and 36 corresponding to HgHMW and Hg-BAL compounds, respectively (Fig. 2C). The gel chromatographic profile of the urine after treatment with mercury and D-PEN showed one peak with fraction 31 corresponding to Hg-D-PEN compound (Fig. 2D). From further characterization of the mercury-binding compound in the urine by paper chromatography, the distribution pattern of 2°3Hg radioactivity detected on the chromatogram of urine after treatment with BGD, BAL, or D-PEN corresponded to that of Hg-GSH and Hg-BGD, Hg-GSH, or Hg-D-PEN compound, respectively (Fig. 3A-C). Furthermore, the characterization of mercury in the bile after treatment with HgC12 and the chelating agents was investigated by the gel chromatographic method (Fig. 4). The Sephadex G-75 (Fig. 4A1) and G-25 (Fig. 4A2) A (Hg alone) 20
20-
Bd
:tYL____ i
i
!
i
i
B (Hg-BGD) Hg-GSH Bd
,o-2j
Hg-BGD
5-
10 20'
C (Hg-BAL)
20
30
1'0
25. D (Hg-D-PEN) Bd
Hg-~SH
Hg-~SH
15"
0
40
Hg-BAL
10'
20
30
40
Hg-GSH
15.
- ~
10.
5'
5.
0
0. 10
2'0
3'0
40
0
1.0
2'0
3'0
4~
Fractionnumber Fig. 4. Gel chromatographic profiles of rat bile after treatment with mercury and chelating agents. Rats were injected i.p. with 2°3HGC12(300/~g Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, the rats were injected i.p. with BGD, BAL, or D-PEN (a quarter of an LD50) and bile samples were collected for 1 h. The bile samples we/'e fractionated on Sephadex G-75 column (I.5 x 16 era) (A1 and B) or G-25 column (1.5 x 39 cm) (A2, C and D). Bd, blue dextran. GSH, glutathione.
164
chromatographic profiles of the bile after administration of HgCI 2 alone showed two peaks with fractions 7 and 14 on G-75 and fractions 14 and 25 on G-25, respectively. In Fig. 4A1 and A2, the first small peak corresponded to Hg-HMW compound and the second main peak corresponded to Hg-GSH compound. The gel chromatographic profile of the bile after treatment with HgCI2 and BGD showed three peaks with fractions 7, 14 and 28 corresponding to Hg-HMW, Hg-GSH and A (Hg-BGD) 10(]
Hg-GSH
8~3 6G Hg-BGD 4~ 2G
B (Hg-BAL) IO0o)
.~
Hg-GSH
80-
g 60 ~o
g-
40
o
lF
C (Hg-D-PEN)
8O
Hg-GSH H~-D-PEN
20 0 origin
front
Fig. 5. Paper chromatography of rat bile after treatment with mercury and chelating agents. Rats were injected i.p. with 2°3HGC12(300 #g Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, the rats were injected i.p. with BGD, BAL, or D-PEN (a quarter of an LDs0 ) and bile samples were collected for 1 h. The bile sample was applied to filter paper. Solvent system: BGD, ethanol-0.1 M ammonium formate buffer (pH 7.4) (7:3); BAL, dioxane-0.1 M ammonium formate buffer (pH 7.4) (9:1); D-PEN, 0.1 M ammonium formate buffer (pH 7.4).
165 H g - B G D compounds, respectively (Fig. 4B). The diagram of the bile after administration of HgCI2 and BAL showed three peaks with fractions 14, 25 and 36 corresponding to H g - H M W , H g - G S H and Hg-BAL compounds (Fig. 4C). The gel chromatographic profile of bile after treatment with HgCI2 and D-PEN showed three peaks corresponding to H g - H M W compound (fraction 14), unknown Hg compound (fraction 22) and H g - G S H compound (fraction 25), respectively (Fig. 4D). The paper chromatographic histograms of the bile of rats treated with the chelating agents after mercury injection are shown in Fig. 5. The diagram of bile after administration of HgC12 and B G D showed two mercury compounds corresponding to H g - G S H and H g - B G D compounds (Fig. 5A). The chromatographic profiles of bile of rats injected with BAL or D-PEN after HgC12 treatment also showed the presence of Hg-GSH, respectively (Fig. 5B and C). Table II shows the percentages of mercury compounds in bile and urine of rats injected with chelating agents after HgCI2 treatment. In the bile and urine of rats treated with HgCI2 and BGD, H g - B G D compound was major and H g - G S H and H g - H M W compounds were minor. The percentages of H g - G S H and Hg-BAL compounds in the bile and urine of rats treated with HgC12 and BAL were 90.7 and 7.6% in the bile and 90.5 and 6.2% in the urine, respectively. The percentages of HgG S H and unknown Hg compounds in the bile after HgC12 and D-PEN treated were 48.4 and 39.9%, respectively, but only Hg-D-PEN compound was excreted in the urine. We further investigated the small intestinal reabsorption of mercury compounds from the bile of rats injected with the chelating agents 24 h after HgC12 treatment. TABLE II PERCENTAGES OF MERCURY COMPOUNDS IN BILE AND URINE OF RATS INJECTED WITH CHELATING AGENTS AFTER MERCURY TREATMENT Rats were injected i.p. with 2°3HGC12(300 ~g Hg and 74 kBq of 2°3Hg/kg).Twenty-fourhours later, the rats were injected i.p. with the chelating agents (a quarter of an LDs0). The bile and urine sampleswere collected for 1 and 5 h, respectivelyand fractionated on Sephadex G-75 column for BGD or Sephadex G-25 column for BAL and ~PEN. The values represent the mean ± S.D. (n = 3). Chelating agent
Mercury compound
Percentage of mercury detected Bile
BGD BAL D-PEN
Hg-BGD Hg-GSH Hg-HMW Hg-GSH Hg-BAL Hg-HMW Hg-D-PEN Hg-GSH Unknown Hg Hg-HMW
63.4 ± 27.6 ± 9.1 ± 90.7 ± 7.6 ± 1.7 ± -48.4 ± 39.6 ± 12.0 ±
Urine 11.2 8.5 2.8 1.8 2.7 0.9
82.9 + 3.1 12.3 ± 3.7 4.8 ± 3.8 90.5 ± 3.1 6.2 ± 2.5 3.3 ±2.1 100.0 ± 0.0
13.1 17.7 5.3
GSH, glutathione; HMW, high molecular weight compound.
166 TABLE III IN SITU SMALL COMPLEXES
INTESTINAL
ABSORPTION
OF
MERCURY-CHELATING
AGENTS
Rats were injected i.p. with 2°3HGC12 (300/zg Hg and 74 kBq of 2°3Hg/kg). Twenty-four hours later, the rats were injected i.p. with the chelating agents (a quarter of an LDs0) and bile samples were collected for 1 h. The values represent the mean 4- S.D. (n = 3). N.D., not detected. Hg absorbed (% of dose) Hg-BGD Bile of rat treated with Hg-BGD Hg-BGD compound Hg-GSH compound Hg-BAL Bile of rat treated with Hg-BAL Hg-BAL compound Hg-BAL compound in bile Hg-BAL compound + glycocholic acid Hg-Cys compound Hg-D-PEN Bile of rat treated with Hg-D-PEN Hg-D-PEN compound
0.18 4- 0.05 N.D. N.D. 27.38 9.91 18.72 22.93 2.39
± 5.79 ± 0.61 4- 2.28 4- 4.73 4- 0.58
0.38 4- 0.37 N.D.
GSH, glutathione; Cys, cysteine.
CG GSH
BAL
O
Cy
!
I
I
I
I
I
I
0
5
10
15
20
25
30
Retention time (rain) Fig. 6. HPLC profile of rat bile after treatment with BAL. Rats were injected i.p. with BAL (0.213 mmol/kg) and bile samples were collected for 1 h. Peaks were identified as follows: Cys, cysteine; CG, cysteinylglycine; GSH, glutathione.
167
As shown in Table III, the intestinal reabsorption of mercury from the bile of rats treated with BGD or D-PEN was only 0.18% or 0.38% of the dose, respectively. However, the intestinal reabsorption of mercury from the bile of rats injected with BAL was 27.38% of the dose. Hg-GSH compound, which was characterized as the main mercury compound in the bile of rats injected with BAL, was little absorbed from the intestinal tracts. On the other hand, the intestinal absorption of Hg-BAL compound or Hg-BAL compound in bile collected from the control rat was shown to be 9.91% or 18.72% of the dose, respectively. In addition, the intestinal absorption of mercury from Hg-BAL compound was increased by the addition of glycocholic acid which is the major constituent of bile acids. As shown in Fig. 6, also, the excretion of BAL in the bile of rats injected with BAL was determined by HPLC method. Discussion
The purpose of the present study was to evaluate the effects of BGD, BAL and D-PEN on the excretion of mercury and to elucidate the chemical characterization and intestinal reabsorption of mercury compounds in the bile and urine of rats injected with the chelating agents after HgCI2 treatment. The injection of the chelating agents at 24 h after mercury treatment showed that BGD and BAL enhanced both the biliary and urinary excretions of mercury and that D-PEN enhanced mainly the uriffary excretion of the metal (Table I). The enhancing effect of BGD on the biliary and urinary excretions of mercury was almost the same as that of BAL and much larger than that o f D-PEN. From chemical characterization of the bile and urine of rats after HgC12 injection using both gel and paper chromatographic methods, the mercury was mainly bound to GSH. The major chemical form of mercury in the bile and urine of rats injected with BGD after HgC12 treatment was mainly Hg-BGD compound in both the bile and urine. This finding suggests that BGD enhances mainly the mercury excretion following HgC12 injection based on the formation of Hg-BGD compound. The chemical form of mercury in the bile and urine of rats injected with BAL after HgCI2 treatment was mainly Hg-GSH compound. It has been shown that the stability constant (log k = 41.6) of Hg-GSH compound [18] is much larger than that (log k = 25.74) of Hg-BAL compound [19]. Our results, which examined the difference in the affinity of mercury for GSH and BAL using the gel chromatographic method, also indicated that the chemical forms of mercury (Hg 2÷) in equimolar mixtures of the two thiols were Hg-GSH compound (90% of applied 2°3Hg) and Hg-BAL compound (10% of applied 2°3Hg) (data not shown). These findings suggest that the replacement of the ligands in Hg-BAL and/or Hg-BAL metabolites by GSH would occur in the body and that the mercury in the body is mainly excreted into the bile and urine in the form of Hg-GSH compound. The mercury after HgC12 and D-PEN treatment was excreted mainly via the urine in the form of Hg-D-PEN compound. In the bile of the minor excretory route of mercury after HgC12 and DPEN injection, the mercury was mainly present in the forms of Hg-GSH and unknown Hg compounds.
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It is known that BAL effectively reduces the kidney level of inorganic mercury when used as an antidote but there is an adverse side effect in that redistribution of mercury to other organs occurs simultaneously [20,21]. We also reported that the injection of BAL at 24 h after mercury treatment caused the redistribution of mercury to the heart, lung and brain [14,22]. Therefore, we investigated the intestinal reabsorption of mercury compounds from the bile of rats injected with the chelating agents after HgC12 treatment (Table III). The intestinal reabsorption of mercury from the bile of rats injected with BGD and D-PEN was negligible, but that of rats injected with BAL was considerably large. Although the chemical form of mercury in the bile of rats injected with BAL after HgCI2 treatment was mainly Hg-GSH compound, Hg-GSH compound was little absorbed from the intestine. It is well known that GSH and its conjugates are degraded to cysteinylglycine and cysteine (Cys) by 7-glutamyl transpeptidase (7-GTP) and dipeptidase [23, 24]. The stability constant of Hg-BAL is much lower than that of Hg-GSH and much larger than that of Hg-Cys (log k = 14.21) [25]. In addition, BAL was found in the bile after injection of the chelating agent (Fig. 3). These findings indicate that Hg-GSH compound excreted in the bile is partly degrated to Hg-Cys form by the intestinal membranous "¥-GTP and dipeptidase and then Hg bound Cys is exchanged for BAL. Hg-BAL compound produced can be efficiently reabsorbed from the intestinal tract and the absorption of Hg-BAL compound is strongly stimulated by glycocholic acid which is a constituent of bile. The redistribution of mercury to tissues of rats treated with BAL after HgCI2 injection is considered to be due to the intestinal reabsorption of mercury compounds excreted in the bile. References 1
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