Mechanisms Underlying The Selective Removal Of Copper From Copperbinding Mt By

4.1. Formation of Three Different Complexes Between Copper and TTM In Vitro Depending on the Relative Molar Ratio of TTM to Copper

Tetrathiomolybdate forms different complexes with Cu (Cu2+) in vitro depending on the relative ratio of TTM to Cu, which can be monitored spectroscopically by the change at the characteristic absorbances of 240, 315, and 465 nm. When Cu is present in the form bound to MT (i.e., Cu-MT or Cu,Zn-MT), three different complexes are formed on a gel filtration HPLC column by the HPLC-ICP MS method depending on the relative ratio of TTM to Cu as represented by the assumed structures shown in Fig. 4.

When a TTM solution in 50 mM Tris-HCl (pH 7.4) is added into a Cu,Zn-MT solution to the molar ratio of Mo/Cu less than unity (Mo/Cu<1), the Cu and Zn peaks of MT become eluted faster than the original retention time of MT at 18.4 (MT-I) and 20.0 min (MT-II) on the HPLC-ICP MS (41).

Welding Ymbols
Fig. 4. Schematic formation of three different complexes between copper and TTM depending on the relative ratio of TTM/Cu in metallothionein.

The Mo peak of TTM becomes eluted at the same retention time as those of Cu and Mo from the original retention time of TTM at 20.8 min, suggesting the formation of a new complex between MT and TTM, tentatively named the MT/TTM complex (41).

When TTM is added into a Cu,Zn,Cd-MT solution at the molar ratio of Mo/Cu greater than unity but less than 2 (1<Mo/Cu<2), the Cu and Mo peaks of the Cu/TTM complex become precipitated in the absence of proteins or become eluted together with high-molecular-weight proteins in the presence of a liver supernatant of LEC rats, Zn and Cd being eluted at the original retention time of MT. When a blood plasma or albumin is present instead of a liver supernatant or together with a liver supernatant, the Cu and Mo peaks are eluted at the same retention time as that of albumin, suggesting the formation of a ternary complex among the Cu removed from MT, Mo in TTM, and albumin. The changes in the elution profiles of the three metals suggest the removal of Cu from MT to form the soluble Cu/TTM complex. The Cu/TTM complex is not eluted out from the columns under the present separation conditions in the absence of proteins. The Cu/TTM complex binds readily to proteins and is then eluted out from columns (42,43).

The addition of TTM to a Cu,Zn,Cd-MT solution at the molar ratio of TTM/Cu greater than 2 insolubilized the Cu and Mo by leaving soluble Zn,Cd-MT. The precipitated Cu/TTM complex was named the insoluble Cu/TTM complex (41).

The production of the three different complexes can be explained by the following scheme: The first step is the formation of the metal-sulfur bridges between MT and TTM (i.e., s^-cu-stâ„¢). The second step is the cleavage of SMT-Cu bond by the participation of TTM to form the soluble Cu/ TTM complex. The soluble Cu/TTM complex is ready to bind to proteins with the highest affinity to albumin. The soluble Cu/TTM complex is readily bound to proteins and not eluted from a gel filtration column in the absence of proteins. However, further production of the Cu/TTM complex results in the formation of the polymeric Cu/TTM complex (the insoluble Cu/TTM complex) and it is precipitated in a nonsoluble form. These steps are given schematically in Fig. 4.

Zn,Cd-MT and/or apo-MT released from the MT/TTM complex at the second step can be stabilized in the presence of Zn or Cd ions in the reaction medium by forming Zn,Cd-MT and/or Zn-MT or Cd-MT

whose thiol groups are fully occupied by Zn and/or Cd. Zn,Cd-MT, Zn-MT, and Cd-MT released from the MT/TTM complex in the presence of Zn or Cd ions are eluted as sharp peaks at the corresponding retention times of MT.

4.2. Removal of Copper Accumulating in the Liver of Lec Rats by TTM In Vivo

4.2.1. A Single Intravenous Injection of TTM

A single intravenous injection of TTM into LEC rats removes Cu from the liver in different ways depending on the dose. At the low and middle doses of 2 and 10 mg TTM/kg body weight of LEC rats (Cu accumulating in the liver at the concentration of approximately 200 |g Cu/g liver), the Cu removed from the liver is excreted into the bloodstream and bile together with equimolar Mo. Mo injected in a form of TTM disappears from the bloodstream within 0.5 h, and then the metal appears again in the plasma in a form bound to albumin together with Cu, the molar ratio of Mo/Cu bound to albumin is approximately unity (40,44-48). The Cu removed from the liver appears more in the bile than in the plasma together with equimolar Mo, both Cu and Mo in the bile being not eluted out from a gel filtration HPLC column under the conventional conditions mentioned earlier. As a result, the Cu removed from the liver is effluxed into the bile and plasma at the molar ratio of approx 7/3, and Mo does not remain in the liver at the low dose (45). The results indicate that the MT/TTM complex observed in vitro at a low dose of TTM is detectable also in vivo, suggesting that the Cu/TTM complex is liberated from the MT/TTM complex.

At the high dose of 50 mg TTM/kg body weight, although the Cu removed from the liver is excreted into the bloodstream and bile together with equimolar Mo, the amounts of Cu and Mo do not increase in a dose-dependent manner and both metals are insolubilized in the liver. The insolubilized Cu/TTM complex is resolubilized slowly with time and excreted into the plasma and bile (40).

Tetrathiomolybdate thus removes Cu from Cu-MT in the form of the Cu/TTM complex at any dose, and equimolar Cu and Mo are effluxed from the liver in the form of soluble Cu/TTM complex into the bile and plasma, the relative amount of the Cu/TTM complex effluxed into the bile and plasma being approximately 7/3 at any dose even though the efflux is retarded at a high dose of TTM owing to the insolubilization and resolubilization processes (Section 4.2.2.).

4.2.2. Repeated Intraperitoneal Injections of TTM

Repeated intraperitoneal injections of TTM at a dose of 10 mg/kg body weight for eight consecutive days are sufficient to remove about a half the amount of Cu in the liver and to reduce the concentration of Cu in the liver of LEC rats from approx 240 to 80 |g/g liver (41,49). The remaining Cu in the liver was not bound to MT but insolubilized (41,49); that is, TTM removed Cu from MT completely and excessive amounts of TTM formed the insoluble Cu/TTM complex. The Cu insolubilized in the liver was gradually solubilized within days and excreted into both the bile and bloodstream, maintaining the molar ratio of Mo/Cu of approx 1. The relative excretion of both metals into bile and plasma were approx 70% and 30%, respectively (45). Loading of TTM at a higher dose than the present dose of 10 mg/kg or for a longer period than 10 d even at the dose of 10 mg/kg was toxic because of excessive removal of Cu (Section 4.2.4.).

4.2.3. Selectivity in the Removal of Copper by TTM: Selective to Copper Among Various Metals

Tetrathiomolybdate binds Cu through the Mo-S-Cu bridge and this bridge is specific to the three participating elements. Therefore, although TTM can form a complex with metals other than Cu, such as Zn and Cd, the complex formation between TTM and metals (elements) is selective to Cu in the body. In fact, complexes between TTM and metals other than Cu were not detected in vivo. Excessive TTM that does not participated in the formation of complexes with Cu is hydrolyzed to molybdate, this hydrolysis being facilitated in the liver (44). Thus, TTM is highly selective to Cu among diverse metal ions in the ternary complex.

4.2.4. Selectivity in the Removal of Copper by TTM: Selective to Copper Among Copper Species

Tetrathiomolybdate can remove Cu from free-Cu ions (Cu bound nonspecifically to proteins) and Cu bound to MT (Cu coordinated only with thiol groups). However, TTM cannot remove Cu bound to most of Cu enzymes such as Cu,Zn-superoxide dismutase (SOD1) and ceruloplasmin (CP). Cu bound to SOD1 and CP is sometimes decreased after repeated injections of TTM. However, this is assumed not to be the result of direct removal of Cu from those enzymes, but from their chaperones during the metabolic turnover of their enzymes (i.e., Cu is liberated from degraded Cu enzymes is bound to chaperons before being transferred to newly synthesized apo-enzymes).

Because MT is most abundant among Cu-containing proteins in the liver of LEC rats, TTM domi-nantly removes Cu from MT in vivo in LEC rats. TTM can remove Cu from SOD1 and/or other Cu-containing proteins in LEC rats only after complete removal of Cu from MT. This suggests that an excessive dose of TTM causes a Cu deficiency as the chronic side effect.

The primary translation product of CP, CP1059 is processed to CP1040 in the endoplasmic reticulums (ER), and then trafficked to the Golgi apparatus, where Cu is supplied to give Cu-CP1040, Cu being supplied into the Golgi apparatus by Atox1 and Atp7b, and then glycosylated to the mature form holo-CP and the holo-CP is secreted into the plasma (50-52). In the liver of Wilson's disease patients and its animal model, LEC rats, Cu is not supplied to the Golgi apparatus because of the mutation of the gene encoding Atp7b that is present on the Golgi membrane and responsible to the transport of Cu from the cytosol to the Golgi apparatus. CP is, thus, secreted into the plasma in the form of apo-Cp in Wilson's disease patients and LEC rats (53-57).

4.3. Side Effects of Tetrathiomolybdate

Two types of side effects, acute and chronic effects, are known to occur by treating TTM. The chronic effect is a deficiency of Cu caused by an excessive removal of Cu (Section 4.2.4.), and the acute effect is a toxicity of sulfide caused by the production of sulfide ions by the hydrolysis of TTM. As TTM can sequester both free Cu and Cu bound to MT or chaperones, excessive TTM depletes the Cu that should be supplied for the maturation of Cu enzymes, resulting in the causation of Cu deficiency in the body (39). Therefore, excessive treatment with TTM is fatal and has to be avoided.

Tetrathiomolybdate is susceptible to hydrolysis, especially under acidic conditions, to produce sulfide ions. This means that the hydrolysis is accelerated in the gastric juice as the acute effect.

It was observed that a high dose of TTM (50 mg/kg body weight) caused kidney damage, resulting in the increase of blood urea nitrogen (45). However, the mechanism underlying this acute nephro-toxicity of TTM is not known.

A lower dose and treatment with a sufficient interval via nonoral (parenteral) route are recommended to avoid both types of the side effects.

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