MRP2 and the DMPS- and DMSA-mediated elimination of mercury in TR(-) and control rats exposed to thiol S-conjugates of inorganic mercury

Abstract

Cysteine (Cys) and homocysteine (Hcy)-S-conjugates of inorganic mercury (Hg2+) are transportable species of Hg2+ that are taken up readily by proximal tubular cells. The metal chelators, 2,3-dimercaptopropane-1-sulfonic acid (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA), have been used successfully to extract Hg2+ from these cells, presumably via the multidrug resistance protein (Mrp2). In the current study, we tested the hypothesis that Mrp2 is involved in the DMPS- and DMSA-mediated extraction of Hg2+ following administration of Hg2+ as an S-conjugate of Cys or Hcy. To test this hypothesis, control and TR(-) (Mrp2-deficient) rats were injected with 0.5 micromol/kg HgCl2 (containing 203Hg2+) conjugated to 1.25 micromol/kg Cys or Hcy. After 24 and 28 h, rats were treated with saline or 100 mg/kg DMPS or DMSA. Tissues were harvested 48 h after Hg2+ exposure. The renal and hepatic burden of Hg2+ was greater in saline-injected TR- rats than in corresponding controls. Accordingly, the content of Hg2+ in the urine and feces was less in TR- rats than in controls. Following treatment with DMPS or DMSA, the renal content of Hg2+ in both groups of rats was reduced significantly and the urinary excretion of Hg2+ was increased. In liver, the effect of each chelator appeared to be dependent upon the form in which Hg2+ was administered. In vitro experiments provide direct evidence indicating that DMPS and DMSA-S-conjugates of Hg2+ are substrates for Mrp2. Overall, these data support our hypothesis that Mrp2 is involved in the DMPS and DMSA-mediated extraction of the body burden of Hg2+.

FIG. 1.

Content of Hg²⁺ in the total renal mass (% of administered dose) of control and TR⁻ rats injected (i.v.) with 0.5 μmol/kg HgCl₂ and 1.25 μmol/kg Cys. Rats were injected (i.p.) with 100 mg/kg DMPS, 100 mg/kg DMSA, or 2 ml/kg saline 24 and 28 h after injection of HgCl₂. Kidneys were harvested for determination of Hg²⁺ content 48 h exposure to HgCl₂. Data represent mean ± SE of four rats. *Significantly different (p < 0.05) from the corresponding mean for the saline-injected rats of the same strain. **Significantly different (p < 0.05) from the corresponding mean for each of the other groups of rats of the same strain. +Significantly different (p < 0.05) from the corresponding mean for control rats treated in the same manner.

FIG. 2.

Concentration of Hg²⁺ (% of administered dose/g tissue) in the various zones of kidney of control and TR⁻ rats injected (i.v.) with 0.5 μmol/kg HgCl₂ and 1.25 μmol/kg Cys. Rats were injected (i.p.) with 100 mg/kg DMPS (A), 100 mg/kg DMSA (B), or 2 ml/kg saline 24 and 28 h after injection of HgCl₂. Kidneys were harvested for determination of Hg²⁺ content 48 h exposure to HgCl₂. Data represent mean ± SE of four rats. *Significantly different (p < 0.05) from the corresponding mean for the same strain of rats treated with saline. **Significantly different (p < 0.05) from the corresponding mean for each of the other groups of rats of the same strain.

FIG. 3.

Amount of Hg²⁺ excreted in urine (% of administered dose/24 h) of control and TR⁻ rats injected (i.v.) with 0.5 μmol/kg HgCl₂ and 1.25 μmol/kg Cys. Twenty-four and 28 h later, rats were injected (i.p.) with 100 mg/kg DMPS, 100 mg/kg DMSA, or saline (2 ml/kg). Data represent the amount of Hg²⁺ in urine collected during the 24 h following treatment with DMPS, DMSA, or saline. Data represent mean ± SE of four rats. *Significantly different (p < 0.05) from the corresponding mean for the same strain of rats treated with saline. +Significantly different (p < 0.05) from the corresponding mean for control rats treated in the same manner.

FIG. 4.

Content of Hg²⁺ in feces (% of administered dose/24 h) of control and TR⁻ rats injected (i.v.) with 0.5 μmol/kg HgCl₂ and 1.25 μmol/kg Cys. Rats were injected (i.p.) 24 and 28 h later with 100 mg/kg DMPS, 100 mg/kg DMSA, or 2 ml/kg saline. Data represent the amount of Hg²⁺ in feces collected during the 24 h following treatment with DMPS, DMSA, or saline. *Significantly different (p < 0.05) from the corresponding mean for the same strain of rats treated with saline. +Significantly different (p < 0.5) from the corresponding mean for control rats treated in the same manner.

FIG. 5.

Content of Hg²⁺ in liver (% of administered dose) of control and TR⁻ rats injected (i.v.) with 0.5 μmol/kg HgCl₂ and 1.25 μmol/kg Cys. Rats were injected (i.p.) with 100 mg/kg DMPS, 100 mg/kg DMSA, or 2 ml/kg saline 24 and 28 h after injection of HgCl₂. Kidneys were harvested for determination of Hg²⁺ content 48-h exposure to HgCl₂. Data represent mean ± SE of four rats. *Significantly different (p < 0.05) from the corresponding mean for the same strain of rats treated with saline. **Significantly different (p < 0.05) from the corresponding mean for each of the other groups of rats of the same strain. +Significantly different (p < 0.05) from the corresponding mean for control rats treated in the same manner.

FIG. 6.

Content of Hg²⁺ in the total renal mass (% of administered dose) of control and TR⁻ rats injected (i.v.) with 0.5 μmol/kg HgCl₂ and 1.25 μmol/kg Hcy. Rats were injected (i.p.) with 100 mg/kg DMPS, 100 mg/kg DMSA, or 2 ml/kg saline 24 and 28 h after injection of HgCl₂. Kidneys were harvested for determination of Hg²⁺ content 48 h exposure to HgCl₂. Data represent mean ± SE of four rats. *Significantly different (p < 0.05) from the corresponding mean for the same strain of rats treated with saline. **Significantly different (p < 0.05) from the corresponding mean for each of the other groups of rats of the same strain. +Significantly different (p < 0.05) from the corresponding mean for control rats treated in the same manner.

FIG. 7.

Concentration of Hg²⁺ (% of administered dose/g tissue) in the various zones of kidney of control and TR⁻ rats injected (i.v.) with 0.5 μmol/kg HgCl₂ and 1.25 μmol/kg Hcy. Rats were injected (i.p.) with 100 mg/kg DMPS (A), 100 mg/kg DMSA (B), or 2 ml/kg saline 24 and 28 h after injection of HgCl₂. Kidneys were harvested for determination of Hg²⁺ content 48 h exposure to HgCl₂. Data represent mean ± SE of four rats. *Significantly different (p < 0.05) from the corresponding mean for the same strain of rats treated with saline. **Significantly different (p < 0.05) from the corresponding mean for each of the other groups of rats of the same strain.

FIG. 8.

Amount of Hg²⁺ excreted in urine (% of administered dose/24 h) of control and TR⁻ rats injected (i.v.) with 0.5 μmol/kg HgCl₂ and 1.25 μmol/kg Hcy. Twenty-four and 28 h later, rats were injected (i.p.) with 100 mg/kg DMPS, 100 mg/kg DMSA, or saline (2 ml/kg). Data represent the amount of Hg²⁺ in urine collected during the 24 h following treatment with DMPS, DMSA, or saline. Data represent mean ± SE of four rats. *Significantly different (p < 0.05) from the corresponding mean for the same strain of rats treated with saline. **Significantly different (p < 0.05) from the corresponding mean for each of the other groups of rats of the same strain. +Significantly different (p < 0.05) from the corresponding mean for control rats treated in the same manner.

FIG. 9.

Content of Hg²⁺ in feces (% of administered dose/24 h) of control and TR⁻ rats injected (i.v.) with 0.5 μmol/kg HgCl₂ and 1.25 μmol/kg Hcy. Rats were injected (i.p.) with 100 mg/kg DMPS, 100 mg/kg DMSA, or 2 ml/kg saline 24 and 28 h after the initial injection with HgCl₂. Data represent the amount of Hg²⁺ in feces collected during the 24 h following treatment with DMPS, DMSA, or saline. *Significantly different (p < 0.05) from the corresponding mean for the same strain of rats treated with saline. +Significantly different (p < 0.5) from the corresponding mean for control rats treated in the same manner.

FIG. 10.

Content of Hg²⁺ in liver (% of administered dose) of control and TR⁻ rats injected (i.v.) with 0.5 μmol/kg HgCl₂ and 1.25 μmol/kg Hcy. Rats were injected (i.p.) with 100 mg/kg DMPS, 100 mg/kg DMSA, or 2 ml/kg saline 24 and 28 h after injection of HgCl₂. Kidneys were harvested for determination of Hg²⁺ content 48-h exposure to HgCl₂. Data represent mean ± SE of four rats. *Significantly different (p < 0.05) from the corresponding mean for the same strain of rats treated with saline. **Significantly different (p < 0.05) from the corresponding mean for each of the other groups of rats of the same strain. +Significantly different (p < 0.05) from the corresponding mean for control rats treated in the same manner.

FIG. 11.

Uptake of Hg²⁺ into inside-out membrane vesicles prepared from either, control Sf9 cells or Sf9 cells transfected with human MRP2. Control and MRP2 vesicles were exposed to HgCl₂ (5μM) conjugated to DMPS (12.5μM) or DMSA (12.5μM) for 15 min at 37°C. Data represent two experiments, each performed in triplicate. *Significantly different (p < 0.05) from the corresponding mean for control vesicles treated with the same conjugate.