Metallobiochemistry of ultratrace levels of bismuth in the rat II. Interaction of Bi with tissue, intracellular and molecular components.

Background: Knowledge on Bi metabolism in laboratory animals refers to studies at "extreme" exposures, i.e. pharmacologically relevant high-doses (mg kg b.w.) in relation to its medical use, or infinitesimal doses (pg kgb.w.) concerning radiobiology protection and radiotherapeutic purposes. There are no specific studies on metabolic patterns of environmental exposure doses (ultratrace level, μg kg b.w.), becoming in this context Bi a "heavy metal fallen into oblivion". We previously reported the results of the metabolic fate of ultratrace levels of Bi in the blood of rats [1]. In reference to the same study here we report the results of the retention and tissue binding of Bi with intracellular and molecular components.

Methods: Animals were intraperitoneally injected with 0.8 μg Bi kg b.w. as Bi(NO), alone or in combination with Fe for the radiolabeling of iron proteins. The use of Bi radiotracer allowed the determination of Bi down to pg fg in biological fluids, tissues, subcellular fractions, and biochemical components isolated by differential centrifugation, size exclusion chromatography, solvent extraction, precipitation, immunoprecipitation and dialysis.

Main Findings: At 24 h post injection the kidney contained by far the highest Bi concentration (10 ng g wt.w.) followed by the thymus, spleen, liver, thyroid, trachea, femur, lung, adrenal gland, stomach, duodenum and pancreas (0.1 to 1.3 ng g wt.w.). Brain and testis showed smaller but consistently significant concentrations of the element (0.03 ng g wt.w). Urine was the predominant route of excretion. Intracellularly, liver, kidney, spleen, testis, and brain cytosols displayed the highest percentages (35%-58%) of Bi of homogenates. Liver and testis nuclei were the organelles with the highest Bi content (24 % and 27 %). However, when the recovered Bi of the liver was recorded as percent of total recovered Bi divided by percent of total recovered protein the lysosomes showed the highest relative specific activity than in other fractions. In the brain subcellular fractions Bi was incorporated by neuro-structures with the protein and not lipidic fraction of the myelin retaining 18 % of Bi of the total homogenate. After the liver intra-subcellular fractionation: (i) 65 % of the nuclear Bi was associated with the protein fraction of the nuclear membranes and 35 % with the bulk chromatin bound to non-histone and DNA fractions; (ii) about 50 % of the mitochondrial Bi was associated with inner and outer membranes being the other half recovered in the intramitochondrial matrix; (iii) in microsomes Bi showed a high affinity (close to 90 %) for the membranous components (rough and smooth membranes); (iv) In the liver cytosol three pools of Bi-binding proteins (molecular size > 300 kDa, 70 kDa and 10 kDa) were observed with ferritin and metallothionein-like protein identified as Bi-binding biomolecules. Three similar protein pools were also observed in the kidney cytosol. However, the amount of Bi, calculated in percent of the total cytosolic Bi, were significantly different compared to the corresponding pools of the liver cytosol.

Conclusions: At the best of our knowledge the present paper represents the first in vivo study, on the basis of an environmental toxicology approach, aiming at describing retention and binding of Bi in the rat at tissue, intracellular and molecular levels.