Abstract: Treatment with 2,3-dimercaptosuccinic acid was more effective than N-acetyl-DL-penicillamine and monomercaptosuccinic acid in mobilizing mercury ...
Acta pharmacol. et toxicol. 1978, 42, 248-252
From the Institute of Occupational Health, Box 8149, Oslo-Dep., OSLO I , and the Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet, OSLO I, Norway
Treatment of Methyl Mercury Poisoning in Mice with 2,3-Dimercaptosuccinic Acid and other Complexing Thiols BY Jan Aaseth and Ernst A. H. Friedheim (Received June 7, 1977; Accepted September 13, 1977)
Abstract: Treatment with 2,3-dimercaptosuccinic acid was more effective than N-acetyl-DL-penicillamine and monomercaptosuccinic acid in mobilizing mercury from mice after the injection of methyl mercuric chloride. Dimercaptosuccinic acid treatment started 4 days after the mercury injection and given for 8 days at a dose of 1 mmol SHjkg per day removed more than 3 of the mercury in the brain, while acetylpenicillamine and mercaptosuccinate correspondingly removed less than 4 of the brain deposits. Neither treatment with 2,3dimercaptopropano-1-sulphonatenor with a new thiolated resin, mercaptostarch, mobilized significant amounts of mercury from the brain. Since the toxicity of dimercaptosuccinate seems to be almost as low as that of D-penicillamine this dithiol may provide a potentially useful agent in clinical poisoning due to methyl mercury. Key-words: Methyl mercuric chloride
-
chelating agents - mice.
It is known that BAL (2,3-dimercapto-l-propanol) is of little importance in experimental and clinical cases of methyl mercury poisoning (Swensson & Ulfvarson 1967; Tokuomi 1968). D-penicillamine has been shown to increase the urinary excretion of mercury after exposure to methyl mercury (Bakir et al. 1973; Ischihara et al. 1974). It is reported, however, that certain derivatives of BAL and penicillamine are more effective than the parent compounds. These are 2,3-dimercaptopropano-I-sulphonate (BAL-sulph.) (Gabard 1976a), 2,3-dimercaptosuccinic acid (DMS) (Friedheim & Corvi 1975; Magos 1976) and Nacetyl-DL-penicillamine (NAPA) (Aaseth 1976b). Non-absorbable complexing thiols such as thiolated polystyrene increased the excretion of mercury because biliary excreted metal is bound in the gut (Clarkson et al. 1973). An experimental comparison of the mobilizing
effect of these new agents and principles has not been reported. It is consequently not settled which of the agents is the drug of choice in clinical cases of methyl mercury poisoning. In the present study we have compared the mobilizing effect of orally given BAL-sulph., DMS, NAPA and a complexing resin mercaptostarch (Trimnell et al. 1977). Monomercaptosuccinic acid (MS) was also included in the study in order to compare its chelation potential with that of its dimercapto analogue DMS. Materials and Methods Radioactive methyl mercuric chloride (203 Hg) was purchased from New England Nuclear, Boston, and purified (>99%) as previously described (Norseth & Clarkson 1970; Aaseth 1976b). NAPA and MS were products of Koch-Light Ltd., England, and BAL-sulph. was obtained from Hey1 & Co., Berlin. DMS was
249
METHYL MERCURY POISONING synthesized as previously described (Friedheim & Corvi 1975). Thiolated starch (mercdptostarch), 0.65 mmol SH/g, was obtained from Dr. Trimnell and coworkers at Northern Regional Laboratory, US Department of Agriculture, Illinois, USA. Female mice of NMRI strain, weighing 20+ I g, were given a single intravenous injection of 5 p o l Hg/kg (0.2 ml/mouse) as 203 Hg-labelled methyl mercuric chloride. The mice were housed in metabolism cages as previously described (Aaseth & Norseth 1974). Oral treatment with I mmol SHjkg of NAPA, BAL-sulph., MS or DMS was started 4 days after the mercury injection and continued for 8 days. The thiol was mixed into the food which was quantitatively consumed each day. One additional group of mice was treated with 2% w/w mercaptostarch mixed into the food from the 4th to the 12th day. Counting of radioactivity and calculation of mercury concentrations in organs, in blood and in the whole bodies were performed as described previously (Aaseth & Norseth 1974). The amount of inorganic mercury in the liver and kidneys was determined by the isotopic exchange method (Norseth & Clarkson 1970).
reduced the blood levels to 0.9 nmol Hg/g (range: 0.7-1.1 nmol/g), while the brain mercury after this treatment, 1.5 nmol/g(range: 1.3-1.7 nmol/g),
2
Results A semilogarithmic plot of the body mercury of the controls was approximately linear during the 12-day-period of observation (fig. 1). From the 4th to the 12th day the mean body mercury of the controls decreased from 3.74 t o 2.10 pmol/kg. The treatment with BAL-sulph., NAPA, MS or DMS accelerated the mercury elimination during this period, resulting in a whole body content of 72%, 47%, 28% or 19%, respectively, of the control value on day 12. The mercaptostarch treatment decreased the body mercury to 1.27 pmol/kg (range: 1.06-1.48 pmol/kg) i.e. to 60% of the controls. All the low molecular weight agents (BAL-sulph., NAPA, MS and DMS) increased the urinary excretion of mercury (table 1). In contrast, mercaptostarch caused an increased faecal output of mercury. Table 2 shows that DMS was the most effective agent for removing mercury from the blood, kidneys, liver and brain, reducing the level to about 7%, 8%, 16% and 35%, respectively, of the controls. MS and NAPA were less effective, decreasing the brain mercury to 55-60% of the control level. The treatment with BAL-sulph. did not significantly reduce the brain levels as compared to thecontrols (i.e. P > 0.05 according to WilcoxonMann-Whitney test used here). Mercaptostarch
L
8
6 Days
10
I2
Fig. 1. Effect of 2,3-dimercaptopropano-l-sulphonate (BAL-sulph.), N-acetyl-DL-penicillamine (NAPA), mercaptosuccinic acid (MS) and dimercaptosuccinic acid (DMS) on the mercury retention in mice after an intravenous injection of methyl mercuric chloride. The treatment was started 4 days after the mercury injection, when the animals were divided in groups of 4 mice. Each group received one of the thiols at a daily dose of 1 mmol SH/kg, and an untreated group served as controls. For each group of mice the mean values are given every other day, and at the 4th and the 12th day the experimental ranges are also given. Table 1 Cumulative mercury excretion (nmol per mouse, mean and range) during 12 days in the same mouse groups as in fig. I , and in a group given mercaptostarch (see text). Faeces Controls NAPA BAL-sulph. MS DMS Mercaptostarch
35 (31-39) 41 (3943) 33 (30-36) 25 (2 1-29) 27 (23-31) 57 (52-62)
Urine 15 (1 3-17)
41 (3745) 34 (30-38) 55 (49-6 1) 62 (57-62) 18 (1 2-24)
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JAN AASETH AND ERNST A. H. FRIEDHEIM Table 2
Total mercury concentrations in organs of the same mouse groups as in fig. 1. 4 days after the methyl mercury injection
12 days after the methyl mercury injection No treatment
No treatment
n=4 Liver Kidney Brain Blood
(i.e. controls) n=4
BAL-sulph. n=4
NAPA n=4
MS n=4
DMS n=4
5.0
4.3 (3.8-5 .O) 8.5 (7.5-9.9) 1.6 (1.5-1.8) 1.2 ( 1.&1.4)
2.5 (2.1-2.9) 5.7 (4.3-7.3)
1.6 (1.2-2.3) 2.7 (2.1-3.8) 0.9
0.8 (0.6-1.0) 1.3 (0.8-1.7) 0.6 (0.54.8) 0.1 (0.094.12)
9.7 (8.1-10.7) 27.5 (21.3-30.4) 2.3 (2.C2.5) 4.9 (4.2-5.7)
(3.9-6.0) 15.4 (11.5-19.0) 1.7 (1.62.2) 1.5
(1.1- 1.7)
was not significantly different from the controls. Methyl mercury in the rat is known to release inorganic mercury slowly (Norseth 1971). Table 3 shows that D M S reduced the levels of inorganic mercury in the kidneys and in the liver to about 30% and 50%, respectively, as compared to the organ levels of inorganic mercury of the untreated controls. MS gave a smaller decrease in the kidney level of inorganic mercury, and the inorganic fraction in the liver was not significantly reduced by this treatment.
Discussion The target organ of methyl mercury is the brain Table 3
Inorganic mercury concentration in organs (nmol/g, mean and range) from mice 12 days after an injection of methyl mercuric chloride (5 pmol Hg/kg). During the last 8-day-period treatment was given with 1 mmol SHjkg daily of mercaptosuccinate (MS) or 2,3-dimercaptosuccinate (DMS). No. of animals
Liver
Kidney
Controls
4
MS
4
0.9 (0.7-1.0) 0.7 (0.54.8)
3.0 (2.1-3.8) 1.3
DMS
4
0.5
0.8 (0.6-0.9)
(0.40.5)
(1.1-1.5)
1.1 ( 1.& 1.3)
0.5
(0.4-0.8)
(0.8-1.1)
0.3 (0.2-0.4)
(Berglund et al. 1971; Berlin & Ullberg 1963). Mercury accumulates in the brain during a period of about 4 days after a methyl mercury injection. Elimination of mercury from the brain is accelerated by treatment with NAPA, MS or DMS, the last agent being the most effective. Considering the mode of action of orally administered chelating agents, three possibilities exist: At first, methyl mercury excreted in bile may form a complex with the agent in the gut, this complex being subsequently excreted. Mercaptostarch seems to act by this mechanism since it increases the faecal excretion of mercury. Secondly, a chelating agent may be absorbed as such from the gastrointestinal tract and react with mercury in the blood or in the extracellular space. Being partly absorbed, BAL-sulph. may work by this mechanism in addition to complex formation with mercury in the gut (Gabard 1976b). NAPA, MS and D M S too, appear to be well absorbed since they increase the urinary metal excretion. These agents can therefore act by the second mechanism, but a third mechanism may also contribute to the effect, that is intracellular complex formation. This latter mechanism has been supposed to facilitate the brain-to-blood efflux of mercury during NAPA treatment, since NAPA has been shown to penetrate cellular membranes such as the red cell membrane (Aaseth 1976b). It has nevertheless been shown that even a
25 1
METHYL MERCURY POISONING
presumably non-penetrating agent such as BALsulph. administered in a high dose (2 mmol SH/kg daily for 5 days) can mobilize 20-25% of the brain mercury. Apparently there is an exchangeable fraction of methyl mercury in brain cells, although at least 75% of the brain mercury is tightly attached to proteins (Berlin et al. 1975; Yoshino et al. 1966). In the present study the DMS treatment removed more than $ of the brain mercury. If this effect should be obtained by extracellular complexation, an additional mechanism must be postulated in the cells to convert protein-bound mercury rapidly into an exchangeable form. The superiority of DMS as compared to NAPA is assumed to reflect suitable pharmacokinetic properties (e.g. absorption, penetration across cellular membranes, or renal clearance of the formed metal complexes) rather than a higher conditional stability constant toward CH,Hg+ (Catsch & Harmuth Hoene 1975). In vitro studies have shown that NAPA and DMS remove approximately equivalentamountsofCH,Hg+ from the CH,Hg+albuminmercaptide (Aaseth, unpublished results). With regard to bifunctional mercury (Hg”), however, the stability constants of dithiols such as DMS are supposed to be higher than those of the corresponding monothiols, as demonstrated by the higher effectiveness of the dithiol in removing inorganic mercury (Aaseth 1976a). It is known that the parent dithiol BAL also forms mercury complexes of high stability, but its marked toxicity in addition to its tendency to form CH,Hg+ complexes which are redistributed to the brain, makes it unsuitable for clinical use in methyl mercury poisoning. Its LD5O-value in mice is reported to be 90 mg/kg, i.e. about 1.5 mmol SHjkg (Zvirblis & Ellin 1976). The LD50values of D-penicillamine (Friedrich & Zimmermann 1975) and NAPA (Aaseth, unpublished results) are much higher, i.e. about 25 mmol SH/kg. LD50 for DMS in mice is in excess of 3 g/kg (( = 30 mmol SH/kg). Ten mmol SH/kg daily for one week of DMS does not give rise to toxic symptoms, while MS in contrast causes 50% mortality (Aaseth, unpublished results). The lower toxicity of DMS as compared to BAL may be related to lower extent of metabolic breakdown in mammalian tissues. The metabolism of DMS and
MS has not yet been studied. Such studies are further motivated by the clinical use of MS as its gold complex (“Myocrisin”) in rheumatoid arthritis. The high effectivity of DMS in combination with its low toxicity in mice, indicates that this agent may provide a clinically useful drug in methyl mercury poisoning. Acknowledgements The author is indebted to Dr. T. Norseth and Dr. E. Jellum for helpful discussion. Dr. D. Trimnell at Northern Regional Laboratories, U.S. Department of Agriculture, Illinois, has kindly donated the mercaptostarch. The technical assistance of Mrs. Sissel Gustafsson is gratefully acknowledged.
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