ABC Efflux Transporters and Solute Carriers in the Early Developing Brain of a Marsupial Monodelphis domestica (South American Gray Short-Tailed Opossum).

This study used a marsupial Monodelphis domestica, which is born very immature and most of its development is postnatal without placental protection. RNA-sequencing (RNA-Seq) was used to identify the expression of influx and efflux transporters (ATP-binding cassettes [ABCs] and solute carriers [SLCs]) and metabolizing enzymes in brains of newborn to juvenile Monodelphis. Results were compared to published data in the developing eutherian rat. To test the functionality of these transporters at similar ages, the entry of paracetamol (acetaminophen) into the brain and cerebrospinal fluid (CSF) was measured using liquid scintillation counting following a single administration of the drug along with its radiolabelled tracer [3H]. Drug permeability studies found that in Monodelphis, brain entry of paracetamol was already restricted at P5; it decreased further in the first week of life and then remained stable until the oldest age group tested (P110). Transcriptomic analysis of Monodelphis brain showed that expression of transporters and their metabolizing enzymes in early postnatal (P) pups (P0, P5, and P8) was relatively similar, but by P109, many more transcripts were identified. When transcriptomes of newborn Monodelphis brain and E19 rat brain and placenta were compared, several transporters present in the rat placenta were also found in the newborn Monodelphis brain. These were absent from E19 rat brain but were present in the adult rat brain. These data indicate that despite its extreme immaturity, the newborn Monodelphis brain may compensate for the lack of placental protection during early brain development by upregulating protective mechanisms, which in eutherian animals are instead present in the placenta.

Development of an Evaluation System Using Intestinal Organoids for Drug Efflux Transport Analysis by an Imaging Approach.

There are several in vitro systems that enable evaluation of the absorption direction, but there are few quantitative systems that enable easy evaluation of the excretion direction. Enteroids, organoids derived from intestine, have been frozen and passaged for various research. But it is not clear how the freezing and passaging affect the expression and function of transporters. We investigated the effects of passage and cryopreservation of enteroids. We focused on P-gp (P-glycoprotein) and compared the transfer rates of rhodamine 123 (Rh123) into the lumen of enteroids with and without a P-gp inhibitor. mRNA expression levels did not change significantly before and after passage and cryopreservation. Accumulation of Rh123 in the lumen of enteroids was observed. With some P-gp inhibitors, excretion of Rh123 into the lumen of enteroids was inhibited and the nonexcreted Rh123 accumulated in enteroids epithelial cells. The transfer rate of Rh123 into the lumen of enteroids with a P-gp inhibitor was significantly decreased compared to that of without a P-gp inhibitor. Before and after passage and cryopreservation, the transfer rate was almost the same as that of primary cultured enteroids. We succeeded in easily evaluating whether a component is a substrate of P-gp using enteroids.

An Integrated Pipeline and Overexpression of a Novel Efflux Transporter, YoeA, Significantly Increases Plipastatin Production in Bacillus subtilis.

Plipastatin, an antimicrobial peptide produced by Bacillus subtilis, exhibits remarkable antimicrobial activity against a diverse range of pathogenic bacteria and fungi. However, the practical application of plipastatin has been significantly hampered by its low yield in wild Bacillus species. Here, the native promoters of both the plipastatin operon and the sfp gene in the mono-producing strain M-24 were replaced by the constitutive promoter P43, resulting in plipastatin titers being increased by 27% (607 mg/mL) and 50% (717 mg/mL), respectively. Overexpression of long chain fatty acid coenzyme A ligase (LCFA) increased the yield of plipastatin by 105% (980 mg/mL). A new efflux transporter, YoeA, was identified as a MATE (multidrug and toxic compound extrusion) family member, overexpression of yoeA enhanced plipastatin production to 1233 mg/mL, an increase of 157%, and knockout of yoeA decreased plipastatin production by 70%; in contrast, overexpression or knockout of yoeA in mono-producing surfactin and iturin engineered strains only slightly affected their production, demonstrating that YoeA acts as the major exporter for plipastatin. Co-overexpression of lcfA and yoeA improved plipastatin production to 1890 mg/mL, which was further elevated to 2060 mg/mL after abrB gene deletion. Lastly, the use of optimized culture medium achieved 2514 mg/mL plipastatin production, which was 5.26-fold higher than that of the initial strain. These results suggest that multiple strain engineering is an effective strategy for increasing lipopeptide production, and identification of the novel transport efflux protein YoeA provides new insights into the regulation and industrial application of plipastatin.

A plasma membrane-localized transporter remobilizes aleurone layer magnesium for seed germination in rice.

The aleurone layer in cereal grains acts as a major reservoir of essential mineral nutrients, significantly influencing seed germination. However, the molecular mechanism underlying the redistribution of nutrients from the aleurone layer in the germinating seed is still not well understood. Here, in rice, we identified a plasma membrane (PM) localized magnesium transporter, MAGNESIUM RELEASE TRANSPORTER 3 (MGR3), is critical for seed germination. OsMGR3 is predominantly expressed in the aleurone layer cells of endosperm, facilitating magnesium remobilization during germination. Non-invasive Micro-test Technology assay data demonstrated that the loss-of-function of OsMGR3 restrained magnesium efflux from the aleurone layer. In the embryo/endosperm grafting experiment, we observed that the mutation of OsMGR3 in the aleurone layer suppressed the growth and differentiation of the embryo during germination. Furthermore, magnesium fluorescence imaging revealed the osmgr3 mutant seeds showed impaired exportation of aleurone layer-stored magnesium to the embryo, consequently delaying germination. Importantly, we discovered that disrupting OsMGR3 could inhibit pre-harvest sprouting without affecting rice yield and quality. Therefore, the magnesium efflux transporter OsMGR3 in the aleurone layer represents a promising genetic target for future agronomic trait improvement.

[Evaluation of Solubility and Membrane Permeability of Middle-Molecule Compounds Using Artificial Membranes and Living Cells].

In contrast to small molecules, middle molecules present a promising therapeutic modality owing to their elevated specificity, minimal adverse effects, capacity to target protein-protein interactions, and, unlike antibody-based drugs, their suitability for oral administration and intracellular target engagement. Post-oral administration, the paramount considerations encompass solubility and membrane permeability during the initial phase until the drug attains systemic circulation. Furthermore, penetration of the cell membrane is essential to accessing intracellular targets. We evaluated the solubility and membrane permeability of 965 compounds sourced from middle molecule libraries affiliated with Hokkaido University, Kitasato University, and the University of Tokyo. To gauge membrane permeability, we employed both the parallel artificial membrane permeability assay (PAMPA) and Caco-2 cell monolayers. Notably, while membrane permeability in Caco-2 cells exhibited an approximate threefold increase in comparison to PAMPA measurements, certain compounds demonstrated permeability levels less than one-third of those observed in Caco-2 cells. Recognizing the potential involvement of efflux transporters expressed in Caco-2 cells in these variations, we conducted additional assessments involving directional transport in the presence of a transporter inhibitor. Our findings suggest that nearly 80% of these compounds serve as substrates for efflux transporters. Considering the relevance of intracellular targets, we shifted our focus from membrane permeation to intracellular uptake, conducting simulations tailored to assess cellular uptake.

Understanding the impact of ABCG2 polymorphisms on drug pharmacokinetics: focus on rosuvastatin and allopurinol.

INTRODUCTION: In addition to the well-established understanding of the pharmacogenetics of drug-metabolizing enzymes, there is growing data on the effects of genetic variation in drug transporters, particularly ATP-binding cassette (ABC) transporters. However, the evidence that these genetic variants can be used to predict drug effects and to adjust individual dosing to avoid adverse events is still limited. AREAS COVERED: This review presents a summary of the current literature from the PubMed database as of February 2024 regarding the impact of genetic variants on ABCG2 function and their relevance to the clinical use of the HMG-CoA reductase inhibitor rosuvastatin and the xanthine oxidase inhibitor allopurinol. EXPERT OPINION: Although there are pharmacogenetic guidelines for the ABCG2 missense variant Q141K, there is still some conflicting data regarding the clinical benefits of these recommendations. Some caution appears to be warranted in homozygous ABCG2 Q141K carriers when rosuvastatin is administered at higher doses and such information is already included in the drug label. The benefit of dose adaption to lower possible side effects needs to be evaluated in prospective clinical studies.

Identification of Escherichia coli multidrug resistance transporters involved in anthocyanin biosynthesis.

The anthocyanin compound cyanidin 3-O-glucoside (C3G) is a natural pigment widely used in food and nutraceutical industries. Its microbial synthesis by E. coli is a promising alternative to the traditional extraction methods. However, part of the synthesized C3G accumulates in the cytoplasm, thus potentially causing growth inhibition and product degradation. Therefore, it is necessary to enhance C3G secretion via exploration of native transporters facilitating C3G export. In this study, we report the screening and verification of native multidrug resistance transporters from 40 candidates in E. coli that can improve the extracellular C3G production when using catechin as the substrate. Overexpression of single transporter genes including fsr, yebQ, ynfM, mdlAB, and emrKY were found to increase C3G production by 0.5- to 4.8-fold. Genetic studies indicated that mdlAB and emrKY are vital transporters in the secretion of C3G. Our study reveals a set of new multidrug resistance transporters for the improvement of microbial biosynthesis of C3G and other anthocyanins.

PfPIN5 promotes style elongation by regulating cell length in Primula forbesii Franch.

BACKGROUND AND AIMS: Style dimorphism is one of the polymorphic characteristics of flowers in heterostylous plants, which have two types of flowers: the pin morph, with long styles and shorter anthers, and the thrum morph, with short styles and longer anthers. The formation of dimorphic styles has received attention in the plant world. Previous studies showed that CYP734A50 in Primula determined style length and limited style elongation and that the brassinosteroid metabolic pathway was involved in regulation of style length. However, it is unknown whether there are other factors affecting the style length of Primula. METHODS: Differentially expressed genes highly expressed in pin morph styles were screened based on Primula forbesii transcriptome data. Virus-induced gene silencing was used to silence these genes, and the style length and anatomical changes were observed 20 days after injection. KEY RESULTS: PfPIN5 was highly expressed in pin morph styles. When PfPIN5 was silenced, the style length was shortened in pin and long-homostyle plants by shortening the length of style cells. Moreover, silencing CYP734A50 in thrum morph plants increased the expression level of PfPIN5 significantly, and the style length increased. The results indicated that PfPIN5, an auxin efflux transporter gene, contributed to regulation of style elongation in P. forbesii. CONCLUSIONS: The results implied that the auxin pathway might also be involved in the formation of styles of P. forbesii, providing a new pathway for elucidating the molecular mechanism of style elongation in P. forbesii.

Identification and function characterization of BnaBOR4 genes reveal their potential for Brassica napus cultivation under high boron stress.

Boron (B) is essential for plant growth, but toxic in excess. In several countries, soil toxic B levels are always a severe agricultural problem in arid and semi-arid regions. Phytoremediation of excess B containing soil is still in its infancy, while high B tolerant plants with elevated protein abundance of B efflux transporter were successfully established or explored. Brassica napus (B. napus) is one of the most important oil crops. However, B efflux transporters underlying excess B tolerance in B. napus remain unknown. Here, we reported that in Brassicaceae species, B. napus had four homologous genes of Arabidopsis AtBOR4 , which were renamed BnaBOR4.1, BnaBOR4.2, BnaBOR4.3 and BnaBOR4.4. BnaBOR4.1, BnaBOR4.2 and BnaBOR4.3 showed constitutive expression and BnaBOR4.4 appeared to be a pseudogene. BnaBOR4.2 and BnaBOR4.3 were expressed in inner cell layers and BnaBOR4.1 in the outer cell layer in root tip, and all were expressed in vascular tissue in the mature zone. B efflux activity assays in yeast demonstrated that BnaBOR4.1, BnaBOR4.2 and AtBOR4 but not BnaBOR4.3 had comparable levels of B transport activity. Structure-functional analysis between BnaBOR4.3 and BnaBOR4.2 demonstrated that amino acid residue substitution at position 297 (Ala vs Pro) and 427 (Met vs Leu) is critical for the B transport activity. Mutant BnaBOR4.3M427L partially restored the B efflux activity, and both mutants BnaBOR4.3A297P and BnaBOR4.3A297P&M427L fully restored B efflux activity, indicating that the Pro297 residue is critical for their function. Further validation of BnaBOR4 was accomplished by growing transgenic Arabidopsis plants under high B conditions. Taken together, our study identified two functional B efflux genes BnaBOR4.1 and BnaBOR4.2 in B. napus, and a key amino acid residue proline 297 associated with B efflux activity. This study highlights the potential of BanBOR4 genes for B. napus cultivation under high B stress.

Human brain microvascular endothelial cells release different types of P-glycoprotein-containing extracellular vesicles upon exposure to doxorubicin.

In the brain, the efflux transporter P-glycoprotein (Pgp) is predominantly located on the luminal membrane of microvascular endothelial cells (BMECs) that form the blood-brain barrier. In addition, Pgp is localized in intracellular organelles involved in Pgp traffic and cycling and, by the release of extracellular vesicles (EVs), in intercellular Pgp transfer to cells with low Pgp expression. We recently described that drug exposure of a human BMEC line (hCMEC/D3) induces the release of Pgp-EGFP-containing EVs; however, the nature of the Pgp-enriched vesicles was not characterized. The two main categories of EVs are exosomes and microvesicles, which differ in origin, size, and molecular cargo. In the present study, we performed similar experiments with hCMEC/D3 cells in the absence and presence of doxorubicin and isolated and characterized the EVs released by the cells during the experiments by differential ultracentrifugation with/without subsequent sucrose gradient fractionation of EV pellets, proteomic profiling, EV size analysis, and confocal fluorescence microscopy. Using cocultures of hCMEC/D3 wildtype cells and cells transduced with MDR1-EGFP or monocultures of hCMEC/D3-MDR1-EGFP cells, we found release of both Pgp-enriched exosomes and microvesicles but analysis of the exosomal marker protein Rab7 indicated that doxorubicin increased particularly the release of exosomes. Transfer experiments with isolated EVs demonstrated EV endocytosis by recipient cells. EV release from BMECs in response to anticancer drugs such as doxorubicin likely serves different functions, including non-genetic intercellular transfer of a resistance phenotype to neighboring BMECs and a mechanism of drug extrusion that contributes to brain protection against potentially toxic chemotherapeutic drugs.

Multidrug resistance of Rhizoctonia solani determined by enhanced efflux for fungicides.

Plant pathogens can develop multidrug resistance (MDR) through metabolomic and efflux activities. Although MDR has been observed in the field, its mechanisms are yet to be further studied. MDR in Rhizoctonia solani induced by the uncoupler SYP-14288, which involved efflux transporters including ATP binding cassette (ABC) and major facilitator superfamily (MFS) have been reported in our previous study. To confirm this, corresponding genes of the wild-type R. solani X19 and its derived MDR mutant X19-7 were compared through transcriptomics, RNA-Seq data validation, and heterologous expression. Genes encoding six ABC transporters and seven MFS transporters were identified to be associated with MDR and mostly showed a constitutively higher expression in X19-7 than in X19 regardless of SYP-14288 treatment. Eight ABC transporter-encoding genes and eight MFS transporter-encoding genes were further characterized by transferring into Saccharomyces cerevisiae. The sensitivity of transformants containing either ABC transporter-encoding gene AG1IA_06082 and MFS transporter-encoding gene AG1IA_08645 was significantly decreased in responses to fungicides having various modes of action including SYP-14288, fluazinam, chlorothalonil, and difenoconazole, indicating that these two genes were related to MDR. The roles of two genes were further confirmed by successfully detecting their protein products and high accumulation of SYP-14288 in yeast transformants. Thus, ABC and MFS transporters contributed to the development of MDR in R. solani. The result helps to understand the cause and mechanisms that influence the efficient use of fungicide.

Drug metabolism-related gene ABCA1 augments temozolomide chemoresistance and immune infiltration abundance of M2 macrophages in glioma.

Gliomas are the most prevalent primary tumor in the central nervous system, with an abysmal 5-year survival rate and alarming mortality. The current standard management of glioma is maximum resection of tumors followed by postoperative chemotherapy with temozolomide (TMZ) or radiotherapy. Low chemosensitivity of TMZ in glioma treatment eventuates limited therapeutic efficacy or treatment failure. Hence, overcoming the resistance of glioma to TMZ is a pressing question. Our research centered on identifying the drug metabolism-related genes potentially involved in TMZ-treated resistance of glioma through several bioinformatics datasets and cell experiments. One efflux transporter, ATP-binding cassette transporter subfamily A1 (ABCA1), was discovered with an upregulated expression level and signaled poor clinical outcomes for glioma patients. The transcript level of ABCA1 significantly elevated across the TMZ-resistant glioma cells in contrast with non-resistant cells. Over-expressed ABCA1 restrained the drug activity of TMZ, and ABCA1 knockdown improved the treatment efficacy. Meanwhile, the results of molecular docking between ABCA1 protein and TMZ showed a high binding affinity. Additionally, co-expression and immunological analysis revealed that ABCA1 facilitates the immune infiltration of M2 macrophages in glioma, thereby stimulating tumor growth and aggravating the poor survival of patients. Altogether, we discovered that the ABCA1 transporter was involved in TMZ chemoresistance and the immune infiltration of M2 macrophages in glioma. Treatment with TMZ after ABCA1 knockdown enhances the chemosensitivity, suggesting that inhibition of ABCA1 may be a potential strategy for improving the therapeutic efficacy of gliomas.

Efflux transport proteins of Tetrahymena thermophila play important roles in resistance to perfluorooctane sulfonate exposure.

The biotoxicity of perfluorooctane sulfonate (PFOS) has been a concern. However, the effects of PFOS on Tetrahymena thermophila, a unicellular model organism, remain unclear. This study aimed to investigate the toxicity and detoxification mechanism of PFOS in this protozoan. PFOS did not show prominent toxic effects on T. thermophila. Cell viability of T. thermophila can be concentration-dependently increased by PFOS. PFOS also increased the stability of cell membranes and the activity of lysosomes. However, PFOS inhibited efflux transporter activities. Most of the PFOS amount remained in the culture medium during the culture periods. Only a low amount of PFOS was absorbed by cells, where PFOS molecules were mainly combined with membrane proteins. The expressions of four membrane protein genes involved in transporting xenobiotics were analyzed by real time-PCR. The gene abcg25 was significantly up-regulated. The growth of abcg25 gene knockout protozoans under PFOS treatment was slightly inhibited. However, the amount of PFOS adsorbed by the knockout protozoans showed no significant difference from the Wild-type protozoans. We concluded that the ABCG25 protein might play a key role in preventing PFOS from entering the cell or being exported from the cells to protect T. thermophila against PFOS. However, ABCG25 was not the only membrane protein able to bind with PFOS.

Imaging the impact of blood-brain barrier disruption induced by focused ultrasound on P-glycoprotein function.

The P-glycoprotein (P-gp/ABCB1) is a major efflux transporter which impedes the brain delivery of many drugs across the blood-brain barrier (BBB). Focused ultrasound with microbubbles (FUS) enables BBB disruption, which immediate and delayed impact on P-gp function remains unclear. Positron emission tomography (PET) imaging using the radiolabeled substrate [11C]metoclopramide provides a sensitive and translational method to study P-gp function at the living BBB. A FUS protocol was devised in rats to induce a substantial and targeted disruption of the BBB in the left hemisphere. BBB disruption was confirmed by the Evan's Blue extravasation test or the minimally-invasive contrast-enhanced MRI. The expression of P-gp was measured 24 h or 48 h after FUS using immunostaining and fluorescence microscopy. The brain kinetics of [11C]metoclopramide was studied by PET at baseline, and both immediately or 24 h after FUS, with or without half-maximum P-gp inhibition (tariquidar 1 mg/kg). In each condition (n = 4-5 rats per group), brain exposure of [11C]metoclopramide was estimated as the area-under-the-curve (AUC) in regions corresponding to the sonicated volume in the left hemisphere, and the contralateral volume. Kinetic modeling was performed to estimate the uptake clearance ratio (R1) of [11C]metoclopramide in the sonicated volume relative to the contralateral volume. In the absence of FUS, half-maximum P-gp inhibition increased brain exposure (+135.0 ± 12.9%, p < 0.05) but did not impact R1 (p > 0.05). Immediately after FUS, BBB integrity was selectively disrupted in the left hemisphere without any detectable impact on the brain kinetics of [11C]metoclopramide compared with the baseline group (p > 0.05) or the contralateral volume (p > 0.05). 24 h after FUS, BBB integrity was fully restored while P-gp expression was maximally down-regulated (-45.0 ± 4.5%, p < 0.001) in the sonicated volume. This neither impacted AUC nor R1 in the FUS + 24 h group (p > 0.05). Only when P-gp was inhibited with tariquidar were the brain exposure (+130 ± 70%) and R1(+29.1 ± 15.4%) significantly increased in the FUS + 24 h/tariquidar group, relative to the baseline group (p < 0.001). We conclude that the brain kinetics of [11C]metoclopramide specifically depends on P-gp function rather than BBB integrity. Delayed FUS-induced down-regulation of P-gp function can be detected. Our results suggest that almost complete down-regulation is required to substantially enhance the brain delivery of P-gp substrates.

Crystal structures of multidrug efflux transporters from Burkholderia pseudomallei suggest details of transport mechanism.

BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae, where both are symmetric trimers composed of three "binding"-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.

Diosmetin Maintains Barrier Integrity by Reducing the Expression of ABCG2 in Colonic Epithelial Cells.

Crohn's disease (CD) is a relapsing and chronic inflammatory bowel disease. Recent advances have highlighted that dysfunction of the barrier function formed by a polarized monolayer of columnar epithelial cells plays a crucial role in the pathophysiology of CD. At present, we reported that diosmetin increased cell viability by reducing the levels of TNFα and IL-6 in lipopolysaccharide (LPS)-treated colonic epithelial Caco-2 cells. Meanwhile, diosmetin conferred a direct effect on maintaining barrier integrity by reducing epithelial permeability and increasing the expression of proteins associated with tight junctions, including zonula occludens-l (ZO-1), occludin, and claudin-1, in LPS-treated Caco-2 cells and in 2,4,6-trinitrobenzene sulfonic acid-induced CD mice. Additionally, diosmetin decreased the protein content of adenosine triphosphate-binding cassette efflux transporter G2 (ABCG2) in vitro and in vivo. Over-expression of ABCG2 had an important impact on the epithelial permeability and barrier-related protein levels induced by LPS in Caco-2 cells. At the same time, Ko143, a specific ABCG2 inhibitor, dramatically enhanced the role of diosmetin in ZO-1 and occludin proteins in LPS-treated Caco-2 cells. Mechanically, diosmetin significantly attenuated the role of LPS in the phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB/AKT), and cAMP-response element binding protein (CREB) in Caco-2 cells. The AMPK inhibitor Compound C obviously prevented the effect of diosmetin on ZO-1 and occludin expression in LPS-treated Caco-2 cells. Taken together, the results of this study suggest that AMPK/AKT/CREB-mediated ABCG2 expression plays a crucial role in diosmetin, improving the barrier dysfunction in CD.

ABCG2 plays a central role in the dysregulation of 25-hydrovitamin D in Crohn's disease.

Vitamin D (VD) deficiency, as indicated by the main circulating form of VD metabolite 25-hydrovitamin D3 (25(OH)D3), in patients with Crohn's disease (CD) has been well documented, but the reasons for this remain unclear. In this study, 367 patients with CD and 57 healthy individuals who were enrolled, and the association between 25 (OH)D3 level and clinical biochemical characteristics including hepatic and renal functions, inflammatory response was analyzed with binary logistic regression models. VD metabolic enzymes and transporters were screened with bioinformatical analysis and identified with qRT-PCR and western blot. Compared to the healthy controls, serum 25(OH)D3 was significantly reduced in patients with CD, but the protein level of adenosine triphosphate (ATP)-binding cassette efflux transporter G2 (ABCG2) was evidently increased in the ileum and colon. Meanwhile, in lipopolysaccharide (LPS)-treated CaCO2 cells, the mRNA and protein levels of ABCG2 were significantly increased, and the overexpression of ABCG2 obviously promoted 25(OH)D3 efflux, but, Ko143, an ABCG2 inhibitors, substantially prevented the efflux of 25(OH)D3. In addition, in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD model mice, the ABCG2 protein levels were significantly increased in the ileum, colon, kidney and liver, and administration of Ko143 significantly inhibited the efflux of 25 (OH) D3in vivo. All of these findings suggest that VD deficiency in patients with CD may be associated with an abnormal increase in ABCG2 expression, but not directly implicated in hepatic and renal function, and inflammatory response in patients with CD.

Protective role of the placental efflux transporter BCRP/ABCG2 in the relationship between prenatal cadmium exposure, placenta weight, and size at birth.

BACKGROUND AND AIM: Placental efflux transporter proteins, such as BCRP, reduce the placental and fetal toxicity of environmental contaminants but have received little attention in perinatal environmental epidemiology. Here, we evaluate the potential protective role of BCRP following prenatal exposure to cadmium, a metal that preferentially accumulates in the placenta and adversely impacts fetal growth. We hypothesized that individuals with a reduced function polymorphism in ABCG2, the gene encoding BCRP, would be most vulnerable to the adverse impacts of prenatal cadmium exposure, notably, smaller placental and fetal size. METHODS: We measured cadmium in maternal urine samples at each trimester and in term placentas from UPSIDE-ECHO study participants (NY, USA; n = 269). We fit adjusted multivariable linear regression and generalized estimating equation models to examine log-transformed urinary and placental cadmium concentrations in relation to birthweight, birth length, placental weight, and fetoplacental weight ratio (FPR) and stratified models by ABCG2 Q141K (C421A) genotype. RESULTS: Overall 17% of participants expressed the reduced-function ABCG2 C421A variant (AA or AC). Placental cadmium concentrations were inversely associated with placental weight (ß = -19.55; 95%CI: -37.06, -2.04) and trended towards higher FPR (ß = 0.25; 95%CI: -0.01, 0.52) with stronger associations in 421A variant infants. Notably, higher placental cadmium concentrations in 421A variant infants were associated with reduced placental weight (ß = -49.42; 95%CI: 98.87, 0.03), and higher FPR (ß = 0.85, 95%CI: 0.18, 1.52), while higher urinary cadmium concentration was associated with longer birth length (ß = 0.98; 95%CI: 0.37, 1.59), lower ponderal index (ß = -0.09; 95%CI: 0.15, -0.03), and higher FPR (ß = 0.42; 95%CI: 0.14, 0.71). CONCLUSIONS: Infants with reduced function ABCG2 polymorphisms may be particularly vulnerable to the developmental toxicity of cadmium as well as other xenobiotics that are BCRP substrates. Additional work examining the influence of placental transporters in environmental epidemiology cohorts is warranted.

Effects of whole-brain radiation therapy on the blood-brain barrier in immunocompetent and immunocompromised mouse models.

BACKGROUND: Approximately 20% of all cancer patients will develop brain metastases in their lifespan. The standard of care for patients with multiple brain metastases is whole-brain radiation therapy, which disrupts the blood-brain barrier. Previous studies have shown inflammatory mediators play a role in the radiation-mediated increase in permeability. Our goal was to determine if differential permeability post-radiation occurs between immunocompetent and immunocompromised mice. METHODS: We utilized a commissioned preclinical irradiator to irradiate brains of C57Bl/6J wild-type and athymic nude mice. Acute (3-24 h) effects on blood-brain barrier integrity were evaluated with our in-situ brain perfusion technique and quantitative fluorescent and phosphorescent microscopy. The presence of inflammatory mediators in the brain and serum was determined with a proinflammatory cytokine panel. RESULTS: Blood-brain barrier integrity and efflux transporter activity were altered in the immunocompetent mice 12 h following irradiation without similar observations in the immunocompromised mice. We observed increased TNF-α concentrations in the serum of wild-type mice immediately post-radiation and nude mice 12 h post-radiation. The brain concentration of CXCL1 was also increased in both mouse strains at the 12-h time point. CONCLUSIONS: The immune response plays a role in the magnitude of blood-brain barrier disruption following irradiation in a time- and size-dependent manner.

Reduction in ABCG2 mRNA Expression in Human Immortalised Brain Microvascular Endothelial Cells by Ferric Ammonium Citrate is Mediated by Reactive Oxygen Species and Activation of ERK1/2 Signalling.

PURPOSE: The ATP-binding cassette (ABC) transport protein ABCG2 (also known as breast cancer resistance protein (BCRP)) is expressed at the luminal face of the blood-brain barrier (BBB), where it limits the brain uptake of a number of therapeutic drugs. We recently reported that the ABC efflux transporter P-glycoprotein (P-gp) was downregulated in human immortalised brain endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC). The aim of the present study, therefore, was to assess whether BCRP expression is also affected by FAC and identify any signalling mechanisms involved. METHODS: ABCG2 mRNA was assessed by RT-qPCR. Protein levels of BCRP, phosphorylated extracellular-regulated kinases 1 and 2 (p-ERK1/2) and total ERK 1/2 were assessed by Western blot. Reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate. RESULTS: Treatment of hCMEC/D3 cells with FAC (250 µM, 72 h) significantly reduced ABCG2 mRNA levels (32.2 ± 3.7%) without a concomitant reduction in BCRP protein expression. ABCG2 mRNA levels were restored to control levels when co-treated with the antioxidant N-acetylcysteine (NAC), suggesting the effect of FAC was mediated by a ROS-sensitive pathway. We also found that FAC-treatment was associated with increased levels of p-ERK1/2, suggesting involvement of the ERK1/2 signalling pathway in the observed ABCG2 mRNA downregulation. The ERK1/2 signalling pathway inhibitor U0126 restored p-ERK1/2 levels and partially attenuated the FAC-induced reduction in ABCG2 mRNA. CONCLUSIONS: This study suggests that FAC-induced downregulation of ABCG2 mRNA is driven by ROS and ERK1/2 signalling, mechanisms which may be exploited to modulate BCRP expression at the BBB.