Metallobiochemistry of ultratrace levels of bismuth in the rat I. Metabolic patterns of Bi in the blood.

Background: The number of the applications of bismuth (Bi) is rapidly and remarkably increasing, enhancing the chance to increase the levels to which humans are normally daily exposed. The interest to Bi comes also from the potential of Bi-based nanoparticles (BiNPs) for industrial and biomedical purposes. Like other metal-based NPs used in nanomedicine, BiNPs may release ultratrace amounts of Bi ions when injected. The metabolic fate and toxicity of these ions still needs to be evaluated. At present, knowledge of Bi metabolism in laboratory animals refers almost solely to studies under unnatural "extreme" exposures, i.e. pharmacologically relevant high-doses (up to thousand mg kg) in relation to its medical use, or infinitesimal-doses (pg kg as non-carrier-added Bi radioisotopes) for radiobiology protection, diagnostic and radiotherapeutic purposes. No specific study exists on the "metabolic patterns" in animal models exposed to levels of Bi, i.e. at "environmental dose exposure" that reflect the human daily exposure (μg kg).

Methodology: Rats were intraperitoneally injected with 0.8 μg Bi kg bw as Bi(NO) alone or in combination with Fe for radiolabelling of iron proteins. The use of Bi radiotracers allowed the detection and measurement down to pg fg of the element in the blood biochemical compartments and protein fractions as isolated by differential centrifugation, size exclusion- and ion exchange chromatography, electrophoresis, solvent extraction, precipitation and dialysis.

Results: 24 h after the administration, the blood concentration of Bi was 0.18 ng mL, with a repartition plasma/red blod cells (RBC) in a ratio of 2:1. Elution profiles of plasma from gel filtration on Sephadex G-150 showed four pools of Bi-binder proteins with different molecular sizes (> 300 kDa, 160 kDa, 70 kDa and < 6.5 kDa). In the 70 kDa fraction transferrin and albumin were identified as biomolecule carriers for Bi. In red blood cells, Bi was distributed between cytosol and membranes (ghosts) in a ratio of about 5:1. In the cytosol, low molecular components (LMWC) and the hemoglobin associated the Bi in a ratio of about 1.8:1. In the hemoglobin molecule, Bi was bound to the beta polypeptide chain of the globin. In the ghosts, Bi was detected at more than one site of the protein fraction, with no binding with lipids. Dialysis experiments and the consistently high recovery (80-90 %) of Bi from chromatography of Bi-containing biocomponents suggest that Bi was firmly complexed at physiological pH with a low degree of breaking during the applications of experimental protocols for the isolation of the Bi-biocomplexes. These latter were sensitive to acid buffer pH 5, and to the presence of complexing agents in the dialysis fluid.

Conclusions: On the basis of an environmental biochemical toxicology approach, we have undertaken a study on the metabolic patterns of Bi ions in rats at tissue, subcellular and molecular level with the identification of cellular Bi-binding components. As a first part of the study the present work reports the results concerned with the metabolic fate of ultratrace levels of Bi(NO) in the blood.