Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high‐affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of ≈ 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti‐sequence bilitranslocase antibodies, the protein‐modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.

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The interaction between hexavalent chromium Cr(VI), as K 2 CrO 4 , and standard humic acids (HAs) in bulk solution was studied using three complementary analytical methods: UV-Visible spectroscopy, X-ray absorption spectroscopy and differential pulse stripping voltammetry. The observed UV-Vis and X-ray absorption spectra showed that, under our experimental conditions, HAs did not induce reduction of Cr(VI) to its trivalent chemical form. The interaction between Cr(VI) and HAs has rather led to the formation of Cr(VI)-HAs micelles via supramolecular chemical processes. The reported results could contribute towards explaining the relative persistence of ecotoxic hexavalent chromium in soils.

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders caused by prions. Animal TSE include scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids. Effective management of scrapie in many parts of the world, and of CWD in North American deer population is complicated by the persistence of prions in the environment. After shedding from diseased animals, prions persist in soil, withstanding biotic and abiotic degradation. As soil is a complex, multi-component system of both mineral and organic components, it is important to understand which soil compounds may interact with prions and thus contribute to disease transmission. Several studies have investigated the role of different soil minerals in prion adsorption and infectivity; we focused our attention on the interaction of soil organic components, the humic substances (HS), with recombinant prion protein (recPrP) material. We evaluated the kinetics of recPrP adsorption, providing a structural and biochemical characterization of chemical adducts using different experimental approaches. Here we show that HS act as potent anti-prion agents in prion infected neuronal cells and in the amyloid seeding assays: HS adsorb both recPrP and prions, thus sequestering them from the prion replication process. We interpreted our findings as highly relevant from an environmental point of view, as the adsorption of prions in HS may affect their availability and consequently hinder the environmental transmission of prion diseases in ruminants.

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Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment.

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