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Arsenic (As) and cadmium (Cd), two major carcinogenic heavy metals, enters into human food chain by the consumption of rice or rice-based food products. Both As and Cd disturb plant-nutrient homeostasis and hence, reduces plant growth and crop productivity. In the present study, As/Cd modulated responses were studied in non-basmati (IR-64) and basmati (PB-1) rice varieties, at physiological, biochemical and transcriptional levels. At the seedling stage, PB-1 was found more sensitive than IR-64, in terms of root biomass; however, their shoot phenotype was comparable under As and Cd stress conditions. The ionomic data revealed significant nutrient deficiencies in As/Cd treated-roots. The principal component analysis identified NH 4 + as As-associated key macronutrient; while, NH 4 + /NO 3 - and K + was majorly associated with Cd mediated response, in both IR-64 and PB-1. Using a panel of 21 transporter gene expression, the extent of nutritional deficiency was ranked in the order of PB-1(As)

Ca and P metabolic disorders during the egg-laying period can reduce egg production, impair eggshell quality, and even cause bone problems in hens; however, little is known regarding the capacity of duodenal and nephritic Ca and P absorption. Here, the levels of serum Ca and P metabolic indices and the expression of duodenal and renal Ca and P transporter genes were measured in hens at different egg-laying stages. The Ca, 25-(OH)-VD 3 , and 1,25-(OH) 2 -VD 3 content increased during the peak (43 weeks of age) and late (72 weeks of age) egg-laying periods compared to that during the early (23 weeks of age) egg-laying period; however, there were no differences in Pi levels among the three egg-laying periods. Moreover, duodenal VDR and CaBP-D28k mRNA expression was markedly higher but NPt2b mRNA expression was markedly lower during the peak and late egg-laying periods than during the early egg-laying period. Furthermore, nephritic CaBP-D28k , PMCA1b , and FGFR1 mRNA expression was markedly higher but NPt2a and Cyp24a1 mRNA expression was markedly lower during the peak and late egg-laying periods than during the early egg-laying period. In conclusion, the present study indicated that the increased duodenal and nephritic Ca absorption during the peak and late egg-laying periods may be associated with the VD-VDR pathway, while the decreased P absorption despite relatively stable serum P levels in all three egg-laying stages may associated with osteolysis., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

Transport systems are key processes in every living organism: they allow the entry of all essential nutrients into the cell and its compartments and regulate the intracellular concentrations of metabolites. The transport of cell nutrients represents the first step of many metabolic routes and may also regulate such processes. They are also responsible for reaching the effective intracellular concentration of therapeutic drugs and some mechanisms of resistance and tolerance also depend on them. However, the common techniques used to evaluate the metabolites transport in different cells types are not easy to carry out and require extensive training. In this chapter, we report detailed protocols and tips about the expression of transporters, different activity assays and transporter kinetics determination.

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. K p,uu,brain and K p,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, K p,uu,brain for the BCRP substrates was low. In contrast, K p,uu,CSF for both BCRP substrates was close to unity, resulting in K p,uu,CSF /K p,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the expression profiles, and that CSF is not a suitable surrogate for unbound brain concentrations of BCRP substrates in dogs.

Human oligopeptide transporter 1 (hPepT1) mediates the absorption of dietary peptides and a range of clinically relevant drugs. According to the predicted topological structure, hPepT1 contains multiple asparagine residues in putative N-glycosylation sites. This study investigated the influence of the six putative N-glycosylation sites within the extracellular region between transmembrane domains 9 and 10 on hPepT1 transporter function and expression in HEK-293T cells. Our study confirmed that hPepT1 is N-glycosylated in HEK-293T cells with the glycosylated and fully deglycosylated isoforms exhibiting apparent molecular masses of ∼78 and ∼55 kDa, respectively. Transport uptake of Glycylsarcosine (Gly-sar) by the hPepT1-N562Q variant, but not by other single mutants, was moderately impaired. We also constructed multiple N-glycosylation mutants based on the hPepT1-N562Q mutant by mutagenizing the additional asparagine residues N404Q, N408Q, N439Q, N509Q, and N514Q. Transport function showed a graded decrease as the number of mutagenized residues increased and simultaneous removal of all six asparagine residues essentially abolished transport activity. Kinetic studies indicated that the V max values for Gly-sar transport by low activity mutants were decreased compared to those of wild-type, which suggested that the cell surface expression and/or turnover rate of hPepT1 mutants was impaired; K m values were unchanged in most cases. Using immunoblotting and immunofluorescence, the plasma membrane and total cellular expression of the mutant transporters were decreased in accordance with functional impairments. In summary, we provide the first molecular evidence that hPepT1 is modified by N-glycosylation and that all six asparagine residues in the large extracellular loop between transmembrane domains 9 and 10 are subject to N-glycosylation. This information enhances our understanding of the role of the large extracellular loop in hPepT1 regulation and could facilitate the development of new hPepT1 substrate drugs with improved bioavailability.

The aim of this study was to incorporate an optimized RPMI2650 nasal cell model into a 3D printed model of the nose to test deposition and permeation of drugs intended for use in the nose. The nasal cell model was optimized for barrier properties in terms of permeation marker and mucus production. RT-qPCR was used to determine the xenobiotic transporter gene expression of RPMI 2650 cells in comparison with primary nasal cells. After 14days in culture, the cells were shown to produce mucus, and to express TEER (define) values and sodium fluorescein permeability consistent with values reported for excised human nasal mucosa. In addition, good correlation was found between RPMI 2650 and primary nasal cell transporter expression values. The purpose-built 3D printed model of the nose takes the form of an expansion chamber with inserts for cells and an orifice for insertion of a spray drug delivery device. This model was validated against the FDA glass chamber with cascade impactors that is currently approved for studies of nasal products. No differences were found between the two apparatus. The apparatus including the nasal cell model was used to test a commercial nasal product containing budesonide (Rhinocort, AstraZeneca, Australia). Drug deposition and transport studies on RPMI 2650 were successfully performed. The new 3D printed apparatus that incorporates cells can be used as valid in vitro model to test nasal products in conditions that mimic the delivery from nasal devices in real life conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Increased expression of the ABCB1 gene in cancer cells is usually connected with occurrence of multidrug resistance (MDR) and poor prognosis. However, the correlation between ABCB1 expression and MDR phenotype is difficult to prove in clinical samples. Most of the researchers believe that these difficulties are due to the poor reliability and sensitivity of assays for detection of ABCB1 expression in clinical samples. However, the complexity of P-gp mediated resistance cannot be reduced to the methodical difficulties only. Here, we addressed the question how widely used methods for detection of ABCB1 expression levels could predict its functional activity and thus its contribution to drug resistance in defined conditions in vitro. The ABCB1 expression was assessed at the mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR), and at the protein level by flow cytometry using UIC2 antibody. The ABCB1 function was monitored using a calcein AM accumulation assay. We observed that K562 cells have approximately 320 times higher level of ABCB1 mRNA than HL-60 cells without detectable function. In addition, resistant K562/Dox cells exhibited significantly higher ABCB1 mRNA expression than resistant K562/HHT cells. However, the functional tests clearly indicated opposite results. Flow cytometric assessment of P-gp, although suggested as a reliable method, contradicted the functional test in K562/Dox and K562/HHT cells. We further used a set of MDR cells expressing various levels of P-gp. Similarly here, flow cytometry not always corresponded to the functional analysis. Our results strongly suggest that an approach which exclusively relies on a simple correlation between ABCB1 expression, either at the mRNA level or protein level, and overall resistance may fail to predict actual contribution of P-gp to overall resistance as the data indicating transporter expression reflect its function only roughly even in well-defined in vitro conditions., (© 2014 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd.)

The brain capillary endothelial cells that constitute the blood-brain barrier express multiple ABC transport proteins on the luminal, blood-facing, plasma membrane. These transporters function as ATP-driven efflux pumps for xenobiotics and endogenous metabolites. High expression of these ABC transporters at the barrier is a major obstacle to the delivery of therapeutics, including chemotherapeutics, to the CNS. Here, I review the signals that alter ABC transporter expression and transport function with an emphasis on P-glycoprotein, Mrp2, and breast cancer resistance protein (BCRP), the efflux transporters for which we have the most detailed picture of regulation. Recent work shows that transporter protein expression can be upregulated in response to inflammatory and oxidative stress, therapeutic drugs, diet, and persistent environmental pollutants; as a consequence, drug delivery to the brain is reduced (potentially bad and ugly). In contrast, basal transport activity of P-glycoprotein and BCRP can be reduced through complex signaling pathways that involve events in and on the brain capillary endothelial cells. Targeting these signaling events provides opportunities to rapidly and reversibly increase brain accumulation of drugs that are substrates for the transporters (potentially good). The clinical usefulness of targeting signaling to reduce efflux transporter activity and improve drug delivery to the CNS remains to be established., (© 2015 Elsevier Inc. All rights reserved.)

The metabolic disorder glutaric aciduria type 1 (GA1) is caused by deficiency of the mitochondrial glutaryl-CoA dehydrogenase (GCDH), leading to accumulation of the pathologic metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in blood, urine and tissues. Affected patients are prone to metabolic crises developing during catabolic conditions, with an irreversible destruction of striatal neurons and a subsequent dystonic-dyskinetic movement disorder. The pathogenetic mechanisms mediated by GA and 3OHGA have not been fully characterized. Recently, we have shown that GA and 3OHGA are translocated through membranes via sodium-dependent dicarboxylate cotransporter (NaC) 3, and organic anion transporters (OATs) 1 and 4. Here, we show that induced metabolic crises in Gcdh(-/-) mice lead to an altered renal expression pattern of NaC3 and OATs, and the subsequent intracellular GA and 3OHGA accumulation. Furthermore, OAT1 transporters are mislocalized to the apical membrane during metabolic crises accompanied by a pronounced thinning of proximal tubule brush border membranes. Moreover, mitochondrial swelling and increased excretion of low molecular weight proteins indicate functional tubulopathy. As the data clearly demonstrate renal proximal tubule alterations in this GA1 mouse model during induced metabolic crises, we propose careful evaluation of renal function in GA1 patients, particularly during acute crises. Further studies are needed to investigate if these findings can be confirmed in humans, especially in the long-term outcome of affected patients., (Copyright © 2013 Elsevier B.V. All rights reserved.)