Circulating low-molecular-weight (poly)phenol metabolites in the brain: unveiling in vitro and in vivo blood-brain barrier transport.

Circulating metabolites resulting from colonic metabolism of dietary (poly)phenols are highly abundant in the bloodstream, though still marginally explored, particularly concerning their brain accessibility. Our goal is to disclose (poly)phenol metabolites' blood-brain barrier (BBB) transport, in vivo and in vitro, as well as their role at BBB level. For three selected metabolites, benzene-1,2-diol-3-sulfate/benzene-1,3-diol-2-sulfate (pyrogallol-sulfate - Pyr-sulf), benzene-1,3-diol-6-sulfate (phloroglucinol-sulfate - Phlo-sulf), and phenol-3-sulfate (resorcinol-sulfate - Res-sulf), BBB transport was assessed in human brain microvascular endothelial cells (HBMEC). Their potential in modulating in vitro BBB properties at circulating concentrations was also studied. Metabolites' fate towards the brain, liver, kidney, urine, and blood was disclosed in Wistar rats upon injection. Transport kinetics in HBMEC highlighted different BBB permeability rates, where Pyr-sulf emerged as the most in vitro BBB permeable metabolite. Pyr-sulf was also the most potent regarding BBB properties improvement, namely increased beta(ß)-catenin membrane expression and reduction of zonula occludens-1 membrane gaps. Whereas no differences were observed for transferrin, increased expression of caveolin-1 upon Pyr-sulf and Res-sulf treatments was found. Pyr-sulf was also capable of modulating gene and protein expression of some solute carrier transporters. Notably, each of the injected metabolites exhibited a unique tissue distribution in vivo, with the remarkable ability to almost immediately reach the brain.

1,4-dihydroxy quininib activates ferroptosis pathways in metastatic uveal melanoma and reveals a novel prognostic biomarker signature.

Uveal melanoma (UM) is an ocular cancer, with propensity for lethal liver metastases. When metastatic UM (MUM) occurs, as few as 8% of patients survive beyond two years. Efficacious treatments for MUM are urgently needed. 1,4-dihydroxy quininib, a cysteinyl leukotriene receptor 1 (CysLT1) antagonist, alters UM cancer hallmarks in vitro, ex vivo and in vivo. Here, we investigated the 1,4-dihydroxy quininib mechanism of action and its translational potential in MUM. Proteomic profiling of OMM2.5 cells identified proteins differentially expressed after 1,4-dihydroxy quininib treatment. Glutathione peroxidase 4 (GPX4), glutamate-cysteine ligase modifier subunit (GCLM), heme oxygenase 1 (HO-1) and 4 hydroxynonenal (4-HNE) expression were assessed by immunoblots. Biliverdin, glutathione and lipid hydroperoxide were measured biochemically. Association between the expression of a specific ferroptosis signature and UM patient survival was performed using public databases. Our data revealed that 1,4-dihydroxy quininib modulates the expression of ferroptosis markers in OMM2.5 cells. Biochemical assays validated that GPX4, biliverdin, GCLM, glutathione and lipid hydroperoxide were significantly altered. HO-1 and 4-HNE levels were significantly increased in MUM tumor explants from orthotopic patient-derived xenografts (OPDX). Expression of genes inhibiting ferroptosis is significantly increased in UM patients with chromosome 3 monosomy. We identified IFerr, a novel ferroptosis signature correlating with UM patient survival. Altogether, we demontrated that in MUM cells and tissues, 1,4-dihydroxy quininib modulates key markers that induce ferroptosis, a relatively new type of cell death driven by iron-dependent peroxidation of phospholipids. Furthermore, we showed that high expression of specific genes inhibiting ferroptosis is associated with a worse UM prognosis, thus, the IFerr signature is a potential prognosticator for which patients develop MUM. All in all, ferroptosis has potential as a clinical biomarker and therapeutic target for MUM.

Nanoscale Bilirubin Analysis in Translational Research and Precision Medicine by the Recombinant Protein HUG.

Bilirubin is a toxicological biomarker for hemolysis and liver diseases. The current automated diazo method used in clinical chemistry has limited applicability in rodent models and cannot be used in small animals relevant to toxicology, microphysiological systems, cell cultures, and kinetic studies. Here, we present a versatile fluorometric method for nanoscale analysis of bilirubin based on its highly specific binding to the recombinant bifunctional protein HELP-UnaG (HUG). The assay is sensitive (LoQ = 1.1 nM), accurate (4.5% relative standard error), and remarkably robust, allowing analysis at pH 7.4-9.5, T = 25-37 °C, in various buffers, and in the presence of 0.4-4 mg × L-1 serum albumin or 30% DMSO. It allows repeated measurements of bilirubinemia in murine models and small animals, fostering the 3Rs principle. The assay determines bilirubin in human plasma with a relative standard error of 6.7% at values that correlate and agree with the standard diazo method. Furthermore, it detects differences in human bilirubinemia related to sex and UGT1A1 polymorphisms, thus demonstrating its suitability for the uniform assessment of bilirubin at the nanoscale in translational and precision medicine.

Cyanidin 3-glucoside targets a hepatic bilirubin transporter in rats.

One of the organ-specific functions of the liver is the excretion of bilirubin into the bile. Membrane transport of bilirubin from the blood to the liver is not only an orphan function, because there is no link to the protein/gene units that perform this function, but also a poorly characterised function. The aim of this study was to investigate the pharmacology of bilirubin uptake in the liver of the female Wistar rat to improve basic knowledge in this neglected area of liver physiology. We treated isolated perfused livers of female rats with repeated single-pass, albumin-free bilirubin boli. We monitored both bilirubin and bilirubin glucuronide in perfusion effluent with a bio-fluorometric assay. We tested the ability of nine molecules known as substrates or inhibitors of sinusoidal membrane transporters to inhibit hepatic uptake of bilirubin. We found that cyanidin 3-glucoside and malvidin 3-glucoside were the only molecules that inhibited bilirubin uptake. These dietary anthocyanins resemble bromosulfophthalein (BSP), a substrate of several sinusoidal membrane transporters. The SLCO-specific substrates estradiol-17 beta-glucuronide, pravastatin, and taurocholate inhibited only bilirubin glucuronide uptake. Cyanidin 3-glucoside and taurocholate acted at physiological concentrations. The SLC22-specific substrates indomethacin and ketoprofen were inactive. We demonstrated the existence of a bilirubin-glucuronide transporter inhibited by bilirubin, a fact reported only once in the literature. The data suggest that bilirubin and bilirubin glucuronide are transported to the liver via pharmacologically distinct membrane transport pathways. Some dietary anthocyanins may physiologically modulate the uptake of bilirubin into the liver.

A preliminary study on the role of Piezo1 channels in myokine release from cultured mouse myotubes.

It has long been known that regular physical exercise induces short and long term benefits reducing the risk of cardiovascular disease, diabetes, osteoporosis, cancer and improves sleep quality, cognitive level, mobility, autonomy in enderly. More recent is the evidence on the endocrine role of the contracting skeletal muscle. Exercise triggers the release of miokines, which act in autocrine, paracrine and endocrine ways controlling the activity of muscles but also of other tissues and organs such as adipose tissue, liver, pancreas, bones, and brain. The mechanism of release is still unclear. Neuromuscular electrical stimulation reproduces the beneficial effects of physical activity producing physiological metabolic, cardiovascular, aerobic responses consistent with those induced by exercise. In vitro, Electrical Pulse Stimulations (EPS) of muscle cells elicit cell contraction and mimic miokine release in the external medium. Here we show that, in cultured mouse myotubes, EPS induce contractile activity and the release of the myokine IL-6. Gadolinium highly reduces EPS-induced IL-6 release, suggesting the involvement of mechanical activated ion channels. The chemical activation of mechanosensitive Piezo1 channels with the specific agonist Yoda1 stimulates IL-6 release similarly to EPS, suggesting the involvement of Piezo1 channels in the control of the myokine release. The expression of Piezo1 protein in myotubes was confirmed by the Western blot analysis. To the best of our knowledge, this is the first evidence of a Piezo1-mediated effect in myokine release and suggests a potential translational use of specific Piezo1 agonists for innovative therapeutic treatments reproducing/enhancing the benefits of exercise mediated by myokines.

Polymer Conjugates of Antimicrobial Peptides (AMPs) with d-Amino Acids (d-aa): State of the Art and Future Opportunities.

In recent years, antimicrobial peptides (AMPs) have enjoyed a renaissance, as the world is currently facing an emergency in terms of severe infections that evade antibiotics' treatment. This is due to the increasing emergence and spread of resistance mechanisms. Covalent conjugation with polymers is an interesting strategy to modulate the pharmacokinetic profile of AMPs and enhance their biocompatibility profile. It can also be an effective approach to develop active coatings for medical implants and devices, and to avoid biofilm formation on their surface. In this concise review, we focus on the last 5 years' progress in this area, pertaining in particular to AMPs that contain d-amino acids, as well as their role, and the advantages that may arise from their introduction into AMPs.

Four selected commercial seaweeds: biologically active compounds, antioxidant and cytotoxic properties.

The aim of this research work was to study the chemical characterisation, antioxidant and cytotoxic activity of ethanolic extracts of four commercial algae species Arame, Kombu, Hijiki and Wakame. The highest scavenging activity has been observed in Arame extract. Antioxidant potential of all extracts was in correlation with total phenol content (Arame extract: 319.15 ± 0.56 mg GAE/g d.w) and it was not in correlation with total carotenoids content (Wakame: 75.15 ± 0.20 mg/g). Polyphenols were quantified using LC-MS/MS technique. Baicalein and amentoflavone were identified in higher amount in relation to other phenols. Intracellular antioxidant activity and cytotoxicity of algae extracts were evaluated on the human prostate cancer cell line PC3. Although presented biomolecules in the extracts have demonstrated in vitro antioxidant activity, they did not show a significant effect on PC3 cells. However, this study opens up broad perspective for the further comprehensive investigation of these, commercial, seaweed's biopotential.

Anthocyanins in Colorectal Cancer Prevention. A Systematic Review of the Literature in Search of Molecular Oncotargets.

Background: Colorectal cancer (CRC) is the malignant process that surges in the terminal part of gastrointestinal tract when adenomatous polyps convert to neoplastic cells able to infiltrate the submucosa. Despite the constant progress in applying preventive measures (screening, colonoscopy) and developing new cures (surgical and chemotherapy), CRC is still one of the leading causes of cancer death worldwide. The importance of natural dietary components in CRC prevention has been recognized. Defining the precise role of the diet and its particular molecular moieties in CRC prevention is of constant scientific interest years behind. Anthocyanins (AC), phenolic phytochemicals present in pigmented plants and vegetables, have been reported to have some role in counteracting CRC carcinogenesis. Nonetheless, evidence coming out the pre-clinical, clinical, and epidemiological studies is still controversial. This review is addressing the need to better comprehend the causes of missing data and discrepancies in investigations on the role of dietary AC in modulating CRC carcinogenesis. Methods: We have analyzed the scientific literature, available in PubMed database, according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement methodology for systematic reviews. Subsequently, two selection strategies, with their screening and eligibility criteria, were applied to retain research articles reporting in vitro and in vivo studies aimed at exploring the molecular mechanisms underlying the observed effects of AC in CRC prevention. Results: From the pool of 82 identified publications, we selected 19 articles reporting experimental or observational data on the effect of AC-enriched diets in CRC prevention in humans or murine species. Furthermore, we selected 10 articles reporting about molecular mechanisms of action of pure AC in CRC experimental models. Conclusions: The major outcome of this review is that AC showed essentially no effect in human studies, whereas AC-enriched diets proved to be effective in experimental murine models of CRC. In cell culture tests, AC showed to interfere with cell signaling pathways related to cell growth and differentiation, apoptosis, oxygen stress, and inflammation response. Further molecular characterizations are required to include AC in the panel of disease-modifying agents.

Capsicum annuum var. grossum (Bell Pepper) Inhibits ß-Secretase Activity and ß-Amyloid1-40 Aggregation.

The deposition of amyloid protein as senile plaques is the major signature of Alzheimer's disease (AD). It is produced by the sequential cleavage of the amyloid precursor protein by secretases. Moreover, peppers are noted for their antiaging and cognitive enhancing properties. Thus, in this study, the effects of polyphenol-rich extracts from bell pepper on amyloid production and aggregation in vitro were investigated. Bell pepper (ripe and unripe) was extracted with methanol-1 N HCl (1:1 v/v). Thereafter, the inhibitory potentials of the extracts on ß-secretase and ß-amyloid1-40 aggregation were determined. Phenolic composition of the pepper fruits was further determined by HPLC-DAD (high performance liquid chromatography-diode array detector). There was a dose-dependent inhibition of ß-secretase by the pepper fruits with the ripe fruits (2.17 ± 0.17 µg/L) showing a significantly (P < .05) higher inhibitory effect than the unripe (3.44 ± 0.11 µg/L). Furthermore, Thioflavin-T and transmission electron microscopy analyses revealed that phenolic extracts from pepper fruits (1 and 10 µg/L) could counteract the initial aggregation of Aß1-40, as well as prevent further aggregation preformed fibrils. These inhibitory activities could be attributed to the predominant presence of phenolic constituents in the pepper fruits. It is possible to conclude that bell pepper could be a possible dietary intervention into the management of AD.

Determination of cyanidin 3-glucoside in rat brain, liver and kidneys by UPLC/MS-MS and its application to a short-term pharmacokinetic study.

Anthocyanins exert neuroprotection in various in vitro and in vivo experimental models. However, no details regarding their brain-related pharmacokinetics are so far available to support claims about their direct neuronal bioactivity as well as to design proper formulations of anthocyanin-based products. To gather this missing piece of knowledge, we intravenously administered a bolus of 668 nmol cyanidin 3-glucoside (C3G) in anaesthetized Wistar rats and shortly after (15 s to 20 min) we collected blood, brain, liver, kidneys and urine samples. Extracts thereof were analysed for C3G and its expected metabolites using UPLC/MS-MS. The data enabled to calculate a set of pharmacokinetics parameters. The main finding was the distinctive, rapid distribution of C3G in the brain, with an apparently constant plasma/brain ratio in the physiologically relevant plasma concentration range (19-355 nM). This is the first report that accurately determines the distribution pattern of C3G in the brain, paving the way to the rational design of future tests of neuroprotection by C3G in animal models and humans.

Inhibition of metalloproteinase and proteasome activities in colon cancer cells by citrus peel extracts.

BACKGROUND: Citrus peels are consumed in the form of infusions, candy or wine, based on their well-documented nutritional and medicinal properties. This study sought to investigate the effect of some citrus peels' [grapefruit (Citrus paradisii), orange (Citrus sinensis) and shaddock (Citrus maxima)] extracts on matrix metalloproteinase (MMP) and proteasome activities in primary human colonic tumor (Caco-2) and the metastatic cell lines (LoVo and LoVo/ADR) in a bid to explain the possible mechanism by which the peels could manage/prevent colon cancer. METHODS: The inhibition of MMP and proteasome activities in the cells by the peel extracts, as well as the identification of phenolic compounds using high-performance liquid chromatography with diode-array detection (HPLC-DAD), was determined. RESULTS: Orange peel extracts had the strongest inhibition of MMP in Caco-2 and LoVo cells, while shaddock had the least. Shaddock peel extracts also had the least MMP inhibition in LoVo/ADR lysates. Grapefruit had the least proteasome inhibition in Caco-2 and LoVo lysates, while there was no significant (p>0.05) difference in the proteasome inhibition of the peel extracts in LoVo/ADR lysates. The extracts inhibited proteasome activity in extract-treated cells, and HPLC fingerprinting of the extracts revealed the presence of some phenolic compounds such as quercetin, caffeic acid, kaempferol, catechin and naringin. CONCLUSIONS: The inhibition of MMP and proteasome activities in colon cancer cell lines suggests the potential use of citrus peels as functional food in the management and/or prevention of colon cancer.

Fate of microbial metabolites of dietary polyphenols in rats: is the brain their target destination?

Different polyphenol compounds are ingested when consuming a serving of fruits rich in polyphenols, spanning from one-phenol hydroxybenzoic acid to more complex polymeric compounds. Only a minor quantity of the polyphenols (5-10%) is absorbed. The remainder reaches the colon and is extensively metabolized by gut microbiota to low-molecular weight metabolites. Their subsequent tissue distribution is still undefined, although these microbial metabolites are currently believed to play a role in human health and disease states. To fill this knowledge gap, we performed a pharmacokinetics experiment in which a single bolus of 23 polyphenol microbial metabolites (total 2.7 µmol) was administered intravenously to rats to reliably reproduce a physiological postabsorption situation. Tissues and urine were collected shortly thereafter (15 s to 15 min) and were analyzed by UHPLC-MS/MS to quantitatively track these compounds. Remarkably, the brain was found to be a specific target organ for 10 of the 23 polyphenol metabolites injected, which significantly increased in the treated animals. In most cases, their appearance in the brain was biphasic, with an early wave at 2 min (4 compounds) and a second wave starting at 5 min; at 15 min, 9 compounds were still detectable. Most compounds were excreted into the urine. The concentrations in the brain of the treated animals were compared against those of the control group by Student's t test, with p-values < 0.1 considered to be statistically significant. These findings provide new perspectives for understanding the role of diet on brain chemistry. Our experimental approach has enabled us to obtain rich metabolomics information from a single experiment involving a limited number of animals.

Involvement of mammalian bilitranslocase-like protein(s) in chlorophyll catabolism of Pisum sativum L. tissues.

Putative pea bilin and cyclic tetrapyrrole transporter proteins were identified by means of an antibody raised against a bilirubin-interacting aminoacidic sequence of mammalian bilitranslocase (TC No. 2.A.65.1.1). The immunochemical approach showed the presence of several proteins mostly in leaf microsomal, chloroplast and tonoplast vesicles. In these membrane fractions, electrogenic bromosulfalein transport activity was also monitored, being specifically inhibited by anti-bilitranslocase sequence antibody. Moreover, the inhibition of transport activity in pea leaf chloroplast vesicles, by both the synthetic cyclic tetrapyrrole chlorophyllin and the heme catabolite biliverdin, supports the involvement of some of these proteins in the transport of linear/cyclic tetrapyrroles during chlorophyll metabolism. Immunochemical localization in chloroplast sub-compartments revealed that these putative bilitranslocase-like transporters are restricted to the thylakoids only, suggesting their preferential implication in the uptake of cyclic tetrapyrrolic intermediates from the stroma during chlorophyll biosynthesis. Finally, the presence of a conserved bilin-binding sequence in different proteins (enzymes and transporters) from divergent species is discussed in an evolutionary context.

Screening of fullerene toxicity by hemolysis assay.

Fullerene is a compound formed during carbon burst that has been produced synthetically starting from the 1990s. The spherical shape and the characteristic carbon bonds of this allotrope (C(60)) have made it a suitable molecule for many applications. During the last decade, the low aqueous solubility of this molecule has been improved by chemical functionalization allowing the use of fullerene derivatives in biological fluids. The characterization of the toxicity potential of fullerenes is therefore of growing interest for any biomedical application. Intravenous injection is one of the possible routes of their administrations and therefore red blood cells are among the first targets of fullerene cytotoxicity. Human red blood cells are easily available and separated from plasma. Membrane disruption by toxic compounds is easily detected in red blood cells as release of hemoglobin in the cell medium, which can be assayed spectrophotometrically at λ = 415 nm. Due to the high molar extinction coefficient of hemoglobin, the assay can be performed on a small amount of both red blood cells and the test compounds, which might be available only in small quantities. So, the hemolysis assay is a simple screening test, whose results can guide further investigations on cytotoxicity in more complex experimental models.

Transport and bioactivity of cyanidin 3-glucoside into the vascular endothelium.

Flavonoids are dietary components involved in decreasing oxidative stress in the vascular endothelium and thus the risk of endothelial dysfunction. However, their very low concentrations in plasma place this role in doubt. Thus, a relationship between the effective intracellular concentration of flavonoids and their bioactivity needs to be assessed. This study examined the uptake of physiological concentrations of cyanidin 3-glucoside, a widespread dietary flavonoid, into human vascular endothelial cells. Furthermore, the involvement of the membrane transporter bilitranslocase (TC No. 2.A.65.1.1) as the key underlying molecular mechanism for membrane transport was investigated by using purified anti-sequence antibodies binding at the extracellular domain of the protein. The experimental observations were carried out in isolated plasma membrane vesicles and intact endothelial cells from human endothelial cells (EA.hy926) and on an ischemia-reperfusion model in isolated rat hearts. Cyanidin 3-glucoside was transported via bilitranslocase into endothelial cells, where it acted as a powerful intracellular antioxidant and a cardioprotective agent in the reperfusion phase after ischemia. These findings suggest that dietary flavonoids, despite their limited oral bioavailability and very low postabsorption plasma concentrations, may provide protection against oxidative stress-based cardiovascular diseases. Bilitranslocase, by mediating the cellular uptake of some flavonoids, is thus a key factor in their protective activity on endothelial function.

Bilberry and blueberry anthocyanins act as powerful intracellular antioxidants in mammalian cells.

Berry anthocyanins have pronounced health effects, even though they have a low bioavailability. The common mechanism underlying health protection is believed to relate to antioxidant activity. Berry extracts, chemically characterised for their phenolic content, were prepared from bilberries (Vaccinium myrtillusL.) and blueberries (Vaccinium corymbosumL.); the bilberry extract was further purified to obtain the anthocyanin fraction. The antioxidant activity of each extract was examined at the cellular level. For this purpose a specific assay, known as cellular antioxidant activity assay (CAA), was implemented in different cell lines: human colon cancer (Caco-2), human hepatocarcinoma (HepG2), human endothelial (EA.hy926) and rat vascular smooth muscle (A7r5). Here we show for the first time that anthocyanins had intracellular antioxidant activity if applied at very low concentrations (<1 µg/l; nM range), thereby providing a long-sought rationale for their health protecting effects in spite of their unfavorable pharmacokinetic properties.

Exceptionally fast uptake and metabolism of cyanidin 3-glucoside by rat kidneys and liver.

To asses the hypothesis that anthocyanins are rapidly taken up from the blood into tissues, where they accumulate up to their bioactivity threshold, an intravenous dose of cyanidin 3-glucoside (1) was administered to anaesthetized rats. Cyanidin 3-glucoside (1) and its metabolites were analyzed in the plasma, kidneys, liver, urine, and bile, using last-generation mass spectrometry. Compound 1 was found to rapidly disappear from plasma (t/2=0.36 min). As soon as 15 s after its administration, both 1 and its methylation product, peonidin 3-glucoside (2), were detected in the plasma, kidneys, and liver. At 1 min, both 1 and 2 had almost disappeared from the plasma, but attained their peak concentrations in the kidneys and in the liver. Compound 2 was rapidly excreted both in the bile and in the urine. Three additional methylated metabolites were detected in traces, namely, delphinidin 3-glucoside (3), petunidin 3-glucoside (4), and malvidin 3-glucoside (5). These data contribute to solving the paradox of the high bioactivity of anthocyanins in spite of their apparent low bioavailability.

Acute cardioprotective and cardiotoxic effects of bilberry anthocyanins in ischemia-reperfusion injury: beyond concentration-dependent antioxidant activity.

Despite being reported to reduce the risk of cardiovascular diseases, little is known about acute direct effects of bilberry anthocyanins on whole mammalian heart under ischemia-reperfusion (I-R) conditions. Bilberry anthocyanins were prepared from the ripe bilberries and analyzed using HPLC-DAD. Their antioxidant activity was evaluated by measuring the intrinsic free radical-scavenging capacity and by cellular antioxidant assay (CAA) on endothelial cells, where we quantified the intracellular capacity to inhibit the formation of peroxyl radicals. Experiments on the isolated rat hearts under I-R were carried out according to the Langendorff method. Perfusion with low concentrations of bilberry anthocyanins (0.01-1 mg/L) significantly attenuated the extent of I-R injury as evidenced by decreasing the release rate of LDH, increasing the postischemic coronary flow, and by decreasing the incidence and duration of reperfusion arrhythmias. High concentrations (5-50 mg/L) diminished cardioprotection and show cardiotoxic activity despite having their radical scavenging and intracellular antioxidant capabilities increased in a concentration-dependent manner. This study reveals the biphasic concentration-dependent bioactivity of bilberry anthocyanins under I-R, which results in strong cardioprotective activity in low concentrations and cardiotoxic activity in high concentrations.

Electrogenic bromosulfalein transport in isolated membrane vesicles: implementation in both animal and plant preparations for the study of flavonoid transporters.

Bromosulfalein is an organic anion dye used in the study of a variety of membrane carriers expressed in animal tissues and involved in transport of drugs and metabolites. The spectrophotometric assay of electrogenic bromosulfalein transport in membrane vesicles, isolated from various mammalian organs or tissues, enables to specifically measure the transport activity of bilitranslocase (TCDB 2.A.65.1.1). The latter is a bilirubin- and flavonoid-specific transporter expressed in rat liver, the organ where its function has been best characterized. The spectrophotometric assay of electrogenic bromosulfalein transport requires minimal volumes of membrane vesicles, is completed within 1 min, and, therefore, is a useful tool to screen the transporter spectrum of potential substrates, by testing them as reversible inhibitors of bromosulfalein transport kinetics. Furthermore, the assay enables to study the progress of time-dependent inactivation of bromosulfalein transport, caused by different protein-specific reagents, including specific anti-sequence antibodies. Inactivation can be retarded by the presence of substrates in a concentration-dependent manner, enabling to derive the dissociation constants of the transporter-substrate complex and thus to gain further insight into the transporter structure-function relationship. This assay, implemented in membrane vesicles isolated from plant organs, has paved the way to the discovery of homologues of bilitranslocase in plants.

Oxidative stress-based cytotoxicity of delphinidin and cyanidin in colon cancer cells.

Colorectal cancer is the second most frequent cause of cancer death in the western world. Although the prognosis has improved after the introduction of newer anticancer drugs, the treatment of metastatic colorectal cancer still remains a challenge due to a high percentage of drug-resistant tumor forms. We aimed at testing whether anthocyanidins exerted cytotoxicity in primary (Caco-2) and metastatic (LoVo and LoVo/ADR) colorectal cancer cell lines. Both cyanidin and delphinidin, though neither pelargonidin nor malvidin, were cytotoxic in metastatic cells only. The cell line most sensitive to anthocyanidins was the drug-resistant LoVo/ADR. There, cellular ROS accumulation, inhibition of glutathione reductase, and depletion of glutathione could be observed. This suggests that anthocyanidins may be used as sensitizing agents in metastatic colorectal cancer therapy.